Cardiac hypertrophy factor

ABSTRACT

Isolated CHF, isolated DNA encoding CHF, and recombinant or synthetic methods of preparing CHF are disclosed, These CHF molecules are shown to influence hypertrophic activity and neurological activity. Accordingly, these compounds or their antagonists may be used for treatment of heart failure, arrhythmic disorders, inotropic disorders, and neurological disorders.

This application is a continuation of and makes reference to, and claimsthe benefits available under 35 U.S.C. Section 120 of, U.S. Ser. No.08/233,609, filed 25 Apr. 1994 now U.S. Pat. No. 5,534,615.

FIELD OF THE INVENTION

This application relates to a cardiac hypertrophy factor (also known ascardiotrophin) for modulating cardiac function in the treatment of heartfailure and for modulating neural function in the treatment ofneurological disorders.

BACKGROUND OF THE INVENTION

Heart failure affects approximately three million Americans, developingin about 400,000 each year. It is currently one of the leading admissiondiagnoses in the U.S. Recent advances in the management of acute cardiacdiseases, including acute myocardial infarction, are resulting in anexpanding patient population that will eventually develop chronic heartfailure.

Current therapy for heart failure is primarily directed to usingangiotensin-converting enzyme (ACE) inhibitors and diuretics. Whileprolonging survival in the setting of heart failure, ACE inhibitorsappear to slow the progression towards end-stage heart failure, andsubstantial numbers of patients on ACE inhibitors have functional classIII heart failure. Moreover, ACE inhibitors consistently appear unableto relieve symptoms in more than 60% of heart failure patients andreduce mortality of heart failure only by approximately 15-20%. Hearttransplantation is limited by the availability of donor hearts. Further,with the exception of digoxin, the chronic administration of positiveinotropic agents has not resulted in a useful drug without accompanyingadverse side effects, such as increased arrhythmogenesis, sudden death,or other deleterious side effects related to survival. Thesedeficiencies in current therapy suggest the need for additionaltherapeutic approaches.

A wide body of data suggests that pathological hypertrophy of cardiacmuscle in the setting of heart failure can be deleterious, characterizedby dilation of the ventricular chamber, an increase in walltension/stress, an increase in the length vs. width of cardiac musclecells, and an accompanying decrease in cardiac performance and function.Studies have shown that the activation of physiological or compensatoryhypertrophy can be beneficial in the setting of heart failure. In fact,the effects of ACE inhibitors have been purported not only to unload theheart, but also to inhibit the pathological hypertrophic response thathas been presumed to be linked to the localized renin-angiotensin systemwithin the myocardium.

On a molecular biology level, the heart functions as a syncytium ofmyocytes and surrounding support cells, called non-myocytes. Whilenon-myocytes are primarily fibroblast/mesenchymal cells, they alsoinclude endothelial and smooth muscle cells. Indeed, although myocytesmake up most of the adult myocardial mass, they represent only about 30%of the total cell numbers present in heart. Because of their closerelationship with cardiac myocytes in vivo, non-myocytes are capable ofinfluencing myocyte growth and/or development. This interaction may bemediated directly through cell--cell contact or indirectly viaproduction of a paracrine factor. Such association in vivo is importantsince both non-myocyte numbers and the extracellular matrix with whichthey interact are increased in myocardial hypertrophy and in response toinjury and infarction. These changes are associated with abnormalmyocardial function.

Cardiac myocytes are unable to divide shortly after birth. Furthergrowth occurs through hypertrophy of the individual cells. Cell culturemodels of myocyte hypertrophy have been developed to understand betterthe mechanisms for cardiac myocyte hypertrophy. Simpson et al., Circ.Res., 51: 787-801 (1982); Chien et al., FASEB J., 5: 3037-3046 (1991).Most studies of heart myocytes in culture are designed to minimizecontamination by non-myocytes. See, for example, Simpson and Savion,Cir. Cres., 50: 101-116 (1982); Libby, J. Mol. Cell. Cardiol., 16:803-811 (1984); Iwaki et al., J. Biol. Chem., 265: 13809-13817 (1990).

Shubaita et al., J. Biol. Chem., 265: 20555-20562 (1990) documented theutility of a culture model to identify peptide-derived growth factorssuch as endothelin-1 that can activate a hypertrophic response. Long etal., Cell Regulation, 2: 1081-1095 (1991) investigated the effect of thecardiac non-myocytes on cardiac myocyte growth in culture. Myocytehypertrophic growth was stimulated in high-density cultures withincreased numbers of non-myocytes and in co-cultures with increasednumbers of non-myocytes. This effect of non-myocytes on myocyte sizecould be reproduced by serum-free medium conditioned by non-myocytecultures. The major myocyte growth-promoting activity in the cultureswas heparin binding. The properties of this growth factor were comparedto various growth factors known to be present in myocardium, includingfibroblast growth factor (FGF), platelet derived growth factor (PDGF),tumor necrosis factor-alpha (TNF-α), and transforming growthfactor-beta1 (TGF-β1). The growth factor of Long et al. was found to belarger than these other known growth factors and to have a differentheparin-Sepharose elution profile from that of all these growth factorsexcept PDGF. Further, it was not neutralized by a PDGF-specificantibody. The authors proposed that it defines a paracrine relationshipimportant for cardiac muscle cell growth and development.

Not only is there a need for an improvement in the therapy of heartfailure such as congestive heart failure, but there is also a need tooffer effective treatment for neurological disorders. Neurotrophicfactors such as insulin-like growth factors, nerve growth factor,brain-derived neurotrophic factor, neurotrophin-3, -4, and -5, andciliary neurotrophic factor have been proposed as potential means forenhancing neuronal survival, for example, as a treatment forneurodegenerative diseases such as amyotrophic lateral sclerosis,Alzheimer's disease, stroke, epilepsy, Huntington's disease, Parkinson'sdisease, and peripheral neuropathy. It would be desirable to provide anadditional therapy for this purpose.

Accordingly, it is an object of the present invention to provide animproved therapy for the prevention and/or treatment of heart failuresuch as congestive heart failure, particularly the promotion ofphysiological forms of hypertrophy or inhibition of pathological formsof hypertrophy, and for the prevention and/or treatment of neurologicaldisorders such as peripheral neuropathy.

It is another object to identify a novel group of cardiac hypertrophyfactor polypeptides and antagonists thereto for use in such therapies.

It is yet another object to provide nucleic acid encoding suchpolypeptides and to use this nucleic acid to produce the polypeptides inrecombinant cell culture.

It is a still further object to provide derivatives and modified formsof such polypeptides, including amino acid sequence variants andcovalent derivatives thereof.

It is an additional object to prepare immunogens for raising antibodiesagainst such polypeptides, as well as to obtain antibodies capable ofbinding them.

It is still another object to provide a novel hypertrophy assay that canbe used, for example, in expression cloning and purification of suchpolypeptides, in evaluation of clones isolated from the expressioncloning, and in identification of antagonists to such polypeptides.

These and other objects of the invention will be apparent to theordinarily skilled artisan upon consideration of the specification as awhole.

SUMMARY OF THE INVENTION

An in vitro neonatal rat heart hypertrophy assay has been developed thatallows for expression cloning and protein purification of the cardiachypertrophy factor (CHF) disclosed herein. The assay capacity of 1000single samples a week coupled with the small sample size requirement of100 μL or less has enabled expression cloning and protein purificationthat would have been impossible using the currently published methods.Hence, in one embodiment, the invention provides a method for assaying atest sample for hypertrophic activity comprising:

(a) plating 96-well plates with a suspension of myocytes at a celldensity of about 7.5×10⁴ cells per mL in Dulbecco's modified Eagle'smedium (D-MEM)/F-12 medium comprising insulin, transferrin, andaprotinin;

(b) culturing the cells;

(c) adding the test sample (such as one suspected of containing a CHF)to the cultured cells;

(d) culturing the cells with the test sample; and

(e) determining if the test sample has hypertrophic activity.

Besides the assay, the invention provides isolated CHF polypeptide,excluding rat CHF polypeptide. This CHF polypeptide is preferablysubstantially homogeneous, may be glycosylated or unglycosylated, andmay be selected from the group consisting of the native sequencepolypeptide, a fragment polypeptide, a variant polypeptide, and achimeric polypeptide. Additionally, the CHF polypeptide may be selectedfrom the group consisting of the polypeptide that is isolated from amammal, the polypeptide that is made by recombinant means, and thepolypeptide that is made by synthetic means. Further, this CHFpolypeptide may be selected from the group consisting of the polypeptidethat is human and the polypeptide that is non-immunogenic in a human.

In another aspect, the isolated CHF polypeptide shares at least 75%amino acid sequence identity with the translated CHF sequence shown inFIG. 1. In a further aspect, the polypeptide is the mature human CHFhaving the translated CHF sequence shown in FIG. 5.

In a still further aspect, the invention provides an isolatedpolypeptide encoded by a nucleic acid having a sequence that hybridizesunder moderately stringent conditions to the nucleic acid sequenceprovided in FIG. 1. Preferably, this polypeptide is biologically active.

In another aspect, the invention provides a chimera comprising CHF fusedto a heterologous polypeptide.

In a still further aspect, the invention provides a compositioncomprising biologically active CHF and a pharmaceutically acceptablecarrier or comprising biologically active CHF fused to an immunogenicpolypeptide.

In yet another aspect, the invention provides an isolated antibody thatis capable of binding CHF and a method for detecting CHF in vitro or invivo comprising contacting the antibody with a sample or cell suspectedof containing CHF and detecting if binding has occurred, as with anELISA.

In still another aspect, the invention provides a method for purifyingCHF comprising passing a mixture of CHF over a column to which is boundthe antibodies and recovering the fraction containing CHF.

In other aspects, the invention comprises an isolated nucleic acidmolecule encoding CHF, a vector comprising the nucleic acid molecule,preferably an expression vector comprising the nucleic acid moleculeoperably linked to control sequences recognized by a host celltransformed with the vector, a host cell comprising the nucleic acidmolecule, including mammalian and bacterial host cells, and a method ofusing a nucleic acid molecule encoding CHF to effect the production ofCHF, comprising culturing a host cell comprising the nucleic acidmolecule. Preferably the host cell is transfected to express CHF nucleicacid and the CHF is recovered from the host cell culture, and ifsecreted, recovered from the culture medium.

In additional aspects, the invention provides an isolated nucleic acidmolecule comprising the open reading frame nucleic acid sequence shownin FIG. 1 or FIG. 5. The invention also provides an isolated nucleicacid molecule excluding rat CHF selected from the group consisting of:

(a) a cDNA clone comprising the nucleotide sequence of the coding regionof the CHF gene shown in FIG. 1 or FIG. 5;

(b) a DNA sequence capable of hybridizing under stringent conditions toa clone of (a); and

(c) a genetic variant of any of the DNA sequences of (a) and (b) whichencodes a polypeptide possessing a biological property of a native CHFpolypeptide.

The invention also provides an isolated DNA molecule having a sequencecapable of hybridizing to the DNA sequence provided in FIG. 1 or FIG. 5under moderately stringent conditions, wherein the DNA molecule encodesa biologically active CHF polypeptide, excluding rat CHF.

In yet another aspect, a method is provided of determining the presenceof a CHF nucleic acid molecule in a test sample comprising contactingthe CHF nucleic acid molecule with the test sample and determiningwhether hybridization has occurred, or comprising hybridizing the CHFnucleic acid molecule to a test sample nucleic acid and determining thepresence of CHF nucleic acid.

In still another aspect, the invention provides a method of amplifying anucleic acid test sample comprising priming a nucleic acid polymerasechain reaction in the test sample with the CHF nucleic acid molecule.

In a still further aspect, the invention provides a CHF antagonist and amethod of identifying such antagonist comprising using cell supernatantsas the test sample in the hypertrophy assay as described above andscreening for molecules that antagonize the hypertrophic activity of aCHF demonstrated in such assay.

In a still further aspect, the invention provides a method for treatinga mammal having or at risk for heart failure, an inotropic disorder, oran arrhythmic disorder comprising administering to a mammal in need ofsuch treatment a therapeutically effective amount of a pharmaceuticalcomposition comprising the CHF or a CHF antagonist in a pharmaceuticallyacceptable carrier.

The invention also provides a method for treating a mammal having or atrisk for a neurological disorder comprising administering to a mammal inneed of such treatment a therapeutically effective amount of apharmaceutical composition comprising the CHF in a pharmaceuticallyacceptable carrier.

In additional embodiments, the invention supplies a method ofidentifying a receptor for CHF comprising using labeled CHF, preferablyradiolabeled CHF, in a cellular receptor assay, allowing the CHF to bindto cells, or using the labeled CHF to pan for cells that contain thereceptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict the nucleotide sequence (sense and anti-sensestrands) (SEQ ID NOS: 1 and 2) and deduced amino acid sequence (SEQ IDNO: 3) of a mouse CHF DNA clone. The underlined complementarynucleotides at position 27 show the start of another mouse CHF cloneused to obtain the full-length clone.

FIG. 2 aligns the translated amino acid sequence of the mouse CHF clone(chf.781) (SEQ ID NO: 3) with the amino acid sequence of human ciliaryneurotrophic factor (humcntf) (SEQ ID NO: 4) to show the extent ofsequence identity.

FIG. 3 shows a graph of atrial natriuretic peptide (ANP) release forphenylephrine (standard curve) and transfections into 293 cells in aneonatal cardiac hypertrophy assay.

FIG. 4 shows a graph of survival of live ciliary ganglion neurons(measured by cell count) as a function of either the ciliaryneutrotrophic factor (CNTF) standard (in ng/mL) or the transfected 293conditioned medium (in fraction of assay volume), using a CNTF standard(circles), medium from a CHF DNA transfection of 293 cells (triangles),and medium from a control DNA transfection of 293 cells (squares).

FIGS. 5A and 5B depict the nucleotide sequence (sense and anti-sensestrands) (SEQ ID NOS: 6 and 7) and deduced amino acid sequence (SEQ IDNO: 8) of a human CHF DNA clone.

FIG. 6 aligns the translated amino acid sequence of the human CHF clone(humct1) (SEQ ID NO: 8) with the translated amino acid sequence of themouse CHF clone (chf.781) (SEQ ID NO: 3) to show the extent of sequenceidentity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

In general, the following words or phrases have the indicated definitionwhen used in the description, examples, and claims:

"CHF" (or "cardiac hypertrophy factor" or "cardiotrophin" or"cardiotrophin-1") is defined herein to be any polypeptide sequence thatpossesses at least one biological property (as defined below) of anaturally occurring polypeptide comprising the polypeptide sequence ofFIG. 1 or the human equivalent thereof shown in FIG. 5. It does notinclude the rat homolog of CHF, i.e., CHF from the rat species. Thisdefinition encompasses not only the polypeptide isolated from a nativeCHF source such as murine embryoid bodies described herein or fromanother source, such as another animal species except rat, includinghumans, but also the polypeptide prepared by recombinant or syntheticmethods. It also includes variant forms including functionalderivatives, alleles, isoforms and analogues thereof.

A "CHF fragment" is a portion of a naturally occurring maturefull-length CHF sequence having one or more amino acid residues orcarbohydrate units deleted. The deleted amino acid residue(s) may occuranywhere in the polypeptide, including at either the N-terminal orC-terminal end or internally. The fragment will share at least onebiological property in common with CHF. CHF fragments typically willhave a consecutive sequence of at least 10, 15, 20, 25, 30, or 40 aminoacid residues that are identical to the sequences of the CHF isolatedfrom a mammal including the CHF isolated from murine embryoid bodies orthe human CHF.

"CHF variants" or "CHF sequence variants" as defined herein meanbiologically active CHFs as defined below having less than 100% sequenceidentity with the CHF isolated from recombinant cell culture or frommurine embryoid bodies having the deduced sequence described in FIG. 1,or with the human equivalent described in FIG. 5. Ordinarily, abiologically active CHF variant will have an amino acid sequence havingat least about 70% amino acid sequence identity with the CHF isolatedfrom murine embryoid bodies or the mature human CHF (see FIGS. 1 and 5),preferably at least about 75%, more preferably at least about 80%, stillmore preferably at least about 85%, even more preferably at least about90%, and most preferably at least about 95%.

A "chimeric CHF" is a polypeptide comprising full-length CHF or one ormore fragments thereof fused or bonded to a second protein or one ormore fragments thereof. The chimera will share at least one biologicalproperty in common with CHF. The second protein will typically be acytokine, growth factor, or hormone such as growth hormone, IGF-I, or aneurotrophic factor such as CNTF, nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),neurotrophin-4 (NT-4), neurotrophin-5 (NT-5), etc.

"Isolated CHF", "highly purified CHF" and "substantially homogeneousCHF" are used interchangeably and mean a CHF that has been purified froma CHF source or has been prepared by recombinant or synthetic methodsand is sufficiently free of other peptides or proteins (1) to obtain atleast 15 and preferably 20 amino acid residues of the N-terminal or ofan internal amino acid sequence by using a spinning cup sequenator orthe best commercially available amino acid sequenator marketed or asmodified by published methods as of the filing date of this application,or (2) to homogeneity by SDS-PAGE under non-reducing or reducingconditions using Coomassie blue or, preferably, silver stain.Homogeneity here means less than about 5% contamination with othersource proteins.

"Biological property" when used in conjunction with either "CHF" or"isolated CHF" means having myocardiotrophic, inotropic,anti-arrhythmic, or neurotrophic activity or having an in vivo effectoror antigenic function or activity that is directly or indirectly causedor performed by a CHF (whether in its native or denatured conformation)or a fragment thereof. Effector functions include receptor binding andany carrier binding activity, agonism or antagonism of CHF, especiallytransduction of a proliferative signal including replication, DNAregulatory function, modulation of the biological activity of othergrowth factors, receptor activation, deactivation, up- ordown-regulation, cell growth or differentiation, and the like. However,effector functions do not include possession of an epitope or antigenicsite that is capable of cross-reacting with antibodies raised againstnative CHF.

An "antigenic function" means possession of an epitope or antigenic sitethat is capable of cross-reacting with antibodies raised against thenative CHF whose sequence is shown in FIG. 1 or another mammalian nativeCHF, including the human homolog whose sequence is shown in FIG. 5. Theprincipal antigenic function of a CHF polypeptide is that it binds withan affinity of at least about 10⁶ L/mole to an antibody raised againstCHF isolated from mouse embryoid bodies or a human homolog thereof.Ordinarily, the polypeptide binds with an affinity of at least about 10⁷L/mole. Most preferably, the antigenically active CHF polypeptide is apolypeptide that binds to an antibody raised against CHF having one ofthe above-described effector functions. The antibodies used to define"biologically activity" are rabbit polyclonal antibodies raised byformulating the CHF isolated from recombinant cell culture or embryoidbodies in Freund's complete adjuvant, subcutaneously injecting theformulation, and boosting the immune response by intraperitonealinjection of the formulation until the titer of the anti-CHF antibodyplateaus.

"Biologically active" when used in conjunction with either "CHF" or"isolated CHF" mean a CHF polypeptide that exhibits hypertrophic,inotropic, anti-arrhythmic, or neurotrophic activity or shares aneffector function of CHF isolated from murine embryoid bodies orproduced in recombinant cell culture described herein, and that may (butneed not) in addition possess an antigenic function. One principaleffector function of CHF or CHF polypeptide herein is influencingcardiac growth or hypertrophy activity, as measured, e.g., by atrialnatriuretic peptide (ANP) release or by the myocyte hypertrophy assaydescribed herein using a specific plating medium and plating density,and preferably using crystal violet stain for readout. The desiredfunction of a CHF (or CHF antagonist) is to increase physiological(beneficial) forms of hypertrophy and decrease pathological hypertrophy.In addition, the CHF herein is expected to display anti-arrhythmicfunction by promoting a more normal electrophysiological phenotype.Another principal effector function of CHF or CHF polypeptide herein isstimulating the proliferation of chick ciliary ganglion neurons in anassay for CNTF activity.

Antigenically active CHF is defined as a polypeptide that possesses anantigenic function of CHF and that may (but need not) in additionpossess an effector function.

In preferred embodiments, antigenically active CHF is a polypeptide thatbinds with an affinity of at least about 10⁶ L/mole to an antibodycapable of binding CHF. Ordinarily, the polypeptide binds with anaffinity of at least about 10⁷ L/mole. Isolated antibody capable ofbinding CHF is an antibody that is identified and separated from acomponent of the natural environment in which it may be present. Mostpreferably, the antigenically active CHF is a polypeptide that binds toan antibody capable of binding CHF in its native conformation. CHF inits native conformation is CHF as found in nature that has not beendenatured by chaotropic agents, heat, or other treatment thatsubstantially modifies the three-dimensional structure of CHF asdetermined, for example, by migration on non-reducing, non-denaturingsizing gels. Antibody used in this determination is rabbit polyclonalantibody raised by formulating native CHF from a non-rabbit species inFreund's complete adjuvant, subcutaneously injecting the formulation,and boosting the immune response by intraperitoneal injection of theformulation until the titer of anti-CHF antibody plateaus.

"Percent amino acid sequence identity" with respect to the CHF sequenceis defined herein as the percentage of amino acid residues in thecandidate sequence that are identical with the residues in the CHFsequence isolated from murine embryoid bodies having the deduced aminoacid sequence described in FIG. 1 or the deduced human CHF amino acidseqeunce described in FIG. 5, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the CHF sequence shall beconstrued as affecting sequence identity or homology. Thus, exemplarybiologically active CHF polypeptides considered to have identicalsequences include prepro-CHF, pro-CHF, and mature CHF.

"CHF microsequencing" may be accomplished by any appropriate standardprocedure provided the procedure is sensitive enough. In one suchmethod, highly purified polypeptide obtained from SDS gels or from afinal HPLC step is sequenced directly by automated Edman (phenylisothiocyanate) degradation using a model 470A Applied Biosystemsgas-phase sequencer equipped with a 120A phenylthiohydantoin (PTH) aminoacid analyzer. Additionally, CHF fragments prepared by chemical (e.g.,CNBr, hydroxylamine, or 2-nitro-5-thiocyanobenzoate) or enzymatic (e.g.,trypsin, clostripain, or staphylococcal protease) digestion followed byfragment purification (e.g., HPLC) may be similarly sequenced. PTH aminoacids are analyzed using the ChromPerfect™ data system (JusticeInnovations, Palo Alto, Calif.). Sequence interpretation is performed ona VAX 11/785 Digital Equipment Co. computer as described by Henzel etal., J. Chromatography, 404:41-52 (1987). Optionally, aliquots of HPLCfractions may be electrophoresed on 5-20% SDS-PAGE, electrotransferredto a PVDF membrane (ProBlott, AIB, Foster City, Calif.) and stained withCoomassie Brilliant Blue. Matsurdiara, J. Biol. Chem., 262: 10035-10038(1987). A specific protein identified by the stain is excised from theblot and N-terminal sequencing is carried out with the gas-phasesequenator described above. For internal protein sequences, HPLCfractions are dried under vacuum (SpeedVac), resuspended in appropriatebuffers, and digested with cyanogen bromide, the Lys-specific enzymeLys-C (Wako Chemicals, Richmond, Va.), or Asp-N (Boehringer Mannheim,Indianapolis, Ind.). After digestion, the resultant peptides aresequenced as a mixture or after HPLC resolution on a C4 column developedwith a propanol gradient in 0.1% trifluoroacetic acid (TFA) prior togas-phase sequencing.

"Isolated CHF nucleic acid" is RNA or DNA containing greater than 16 andpreferably 20 or more sequential nucleotide bases that encodesbiologically active CHF or a fragment thereof, is complementary to theRNA or DNA, or hybridizes to the RNA or DNA and remains stably boundunder moderate to stringent conditions. This RNA or DNA is free from atleast one contaminating source nucleic acid with which it is normallyassociated in the natural source and preferably substantially free ofany other mammalian RNA or DNA. The phrase "free from at least onecontaminating source nucleic acid with which it is normally associated"includes the case where the nucleic acid is present in the source ornatural cell but is in a different chromosomal location or is otherwiseflanked by nucleic acid sequences not normally found in the source cell.An example of isolated CHF nucleic acid is RNA or DNA that encodes abiologically active CHF sharing at least 75%, more preferably at least80%, still more preferably at least 85%, even more preferably 90%, andmost preferably 95% sequence identity with the murine CHF or with thehuman CHF.

"Control sequences" when referring to expression means DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, a ribosome binding site, and possibly, other as yet poorlyunderstood sequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

"Operably linked" when referring to nucleic acids means that the nucleicacids are placed in a functional relationship with another nucleic acidsequence. For example, DNA for a presequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, "operably linked" means that the DNAsequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accord with conventionalpractice.

"Exogenous" when referring to an element means a nucleic acid sequencethat is foreign to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is ordinarily notfound.

"Cell," "cell line," and "cell culture" are used interchangeably hereinand such designations include all progeny of a cell or cell line. Thus,for example, terms like "transformants" and "transformed cells" includethe primary subject cell and cultures derived therefrom without regardfor the number of transfers. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. Where distinct designations are intended, it will be clearfrom the context.

"Plasmids" are autonomously replicating circular DNA moleculespossessing independent origins of replication and are designated hereinby a lower case "p" preceded and/or followed by capital letters and/ornumbers. The starting plasmids herein either are commercially available,are publicly available on an unrestricted basis, or can be constructedfrom such available plasmids in accordance with published procedures. Inaddition, other equivalent plasmids are known in the art and will beapparent to the ordinary artisan.

"Restriction enzyme digestion" when referring to DNA means catalyticcleavage of internal phosphodiester bonds of DNA with an enzyme thatacts only at certain locations or sites in the DNA sequence. Suchenzymes are called "restriction endonucleases." Each restrictionendonuclease recognizes a specific DNA sequence called a "restrictionsite" that exhibits two-fold symmetry. The various restriction enzymesused herein are commercially available and their reaction conditions,cofactors, and other requirements as established by the enzyme suppliersare used. Restriction enzymes commonly are designated by abbreviationscomposed of a capital letter followed by other letters representing themicroorganism from which each restriction enzyme originally was obtainedand then a number designating the particular enzyme. In general, about 1μg of plasmid or DNA fragment is used with about 1-2 units of enzyme inabout 20 μL of buffer solution. Appropriate buffers and substrateamounts for particular restriction enzymes are specified by themanufacturer. Incubation for about 1 hour at 37° C. is ordinarily used,but may vary in accordance with the supplier's instructions. Afterincubation, protein or polypeptide is removed by extraction with phenoland chloroform, and the digested nucleic acid is recovered from theaqueous fraction by precipitation with ethanol. Digestion with arestriction enzyme may be followed with bacterial alkaline phosphatasehydrolysis of the terminal 5' phosphates to prevent the tworestriction-cleaved ends of a DNA fragment from "circularizing" orforming a closed loop that would impede insertion of another DNAfragment at the restriction site. Unless otherwise stated, digestion ofplasmids is not followed by 5' terminal dephosphorylation. Proceduresand reagents for dephosphorylation are conventional as described insections 1.56-1.61 of Sambrook et al., Molecular Cloning: A LaboratoryManual (New York: Cold Spring Harbor Laboratory Press, 1989).

"Recovery" or "isolation" of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide or agarose gelby electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see Lawn et al., Nucleic Acids Res., 9:6103-6114 (1981) and Goeddel et al., Nucleic Acids Res., 8: 4057 (1980).

"Southern analysis" or "Southern blotting" is a method by which thepresence of DNA sequences in a restriction endonuclease digest of DNA ora DNA-containing composition is confirmed by hybridization to a known,labeled oligonucleotide or DNA fragment. Southern analysis typicallyinvolves electrophoretic separation of DNA digests on agarose gels,denaturation of the DNA after electrophoretic separation, and transferof the DNA to nitrocellulose, nylon, or another suitable membranesupport for analysis with a radiolabeled, biotinylated, orenzyme-labeled probe as described in sections 9.37-9.52 of Sambrook etal., supra.

"Northern analysis" or "Northern blotting" is a method used to identifyRNA sequences that hybridize to a known probe such as anoligonucleotide, DNA fragment, cDNA or fragment thereof, or RNAfragment. The probe is labeled with a radioisotope such as ³² P, or bybiotinylation, or with an enzyme. The RNA to be analyzed is usuallyelectrophoretically separated on an agarose or polyacrylamide gel,transferred to nitrocellulose, nylon, or other suitable membrane, andhybridized with the probe, using standard techniques well known in theart such as those described in sections 7.39-7.52 of Sambrook et al.,supra.

"Ligation" is the process of forming phosphodiester bonds between twonucleic acid fragments. For ligation of the two fragments, the ends ofthe fragments must be compatible with each other. In some cases, theends will be directly compatible after endonuclease digestion. However,it may be necessary first to convert the staggered ends commonlyproduced after endonuclease digestion to blunt ends to make themcompatible for ligation. For blunting the ends, the DNA is treated in asuitable buffer for at least 15 minutes at 15° C. with about 10 units ofthe Klenow fragment of DNA polymerase I or T4 DNA polymerase in thepresence of the four deoxyribonucleotide triphosphates. The DNA is thenpurified by phenol-chloroform extraction and ethanol precipitation. TheDNA fragments that are to be ligated together are put in solution inabout equimolar amounts. The solution will also contain ATP, ligasebuffer, and a ligase such as T4 DNA ligase at about 10 units per 0.5 μgof DNA. If the DNA is to be ligated into a vector, the vector is firstlinearized by digestion with the appropriate restrictionendonuclease(s). The linearized fragment is then treated with bacterialalkaline phosphatase or calf intestinal phosphatase to preventself-ligation during the ligation step.

"Preparation" of DNA from cells means isolating the plasmid DNA from aculture of the host cells. Commonly used methods for DNA preparation arethe large- and small-scale plasmid preparations described in sections1.25-1.33 of Sambrook et al., supra. After preparation of the DNA, itcan be purified by methods well known in the art such as that describedin section 1.40 of Sambrook et al., supra.

"Oligonucleotides" are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methodssuch as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid-phase techniques such as described in EP 266,032 published 4 May1988, or via deoxynucleoside H-phosphonate intermediates as described byFroehler et al., Nucl. Acids Res., 14: 5399-5407 (1986). Further methodsinclude the polymerase chain reaction defined below and other autoprimermethods and oligonucleotide syntheses on solid supports. All of thesemethods are described in Engels et al., Agnew. Chem. Int. Ed. Engl., 28:716-734 (1989). These methods are used if the entire nucleic acidsequence of the gene is known, or the sequence of the nucleic acidcomplementary to the coding strand is available. Alternatively, if thetarget amino acid sequence is known, one may infer potential nucleicacid sequences using known and preferred coding residues for each aminoacid residue. The oligonucleotides are then purified on polyacrylamidegels.

"Polymerase chain reaction" or "PCR" refers to a procedure or techniquein which minute amounts of a specific piece of nucleic acid, RNA and/orDNA, are amplified as described in U.S. Pat. No. 4,683,195 issued 28Jul. 1987. Generally, sequence information from the ends of the regionof interest or beyond needs to be available, such that oligonucleotideprimers can be designed; these primers will be identical or similar insequence to opposite strands of the template to be amplified. The 5'terminal nucleotides of the two primers may coincide with the ends ofthe amplified material. PCR can be used to amplify specific RNAsequences, specific DNA sequences from total genomic DNA, and cDNAtranscribed from total cellular RNA, bacteriophage or plasmid sequences,etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol.,51: 263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).As used herein, PCR is considered to be one, but not the only, exampleof a nucleic acid polymerase reaction method for amplifying a nucleicacid test sample comprising the use of a known nucleic acid as a primerand a nucleic acid polymerase to amplify or generate a specific piece ofnucleic acid.

"Stringent conditions" are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015M NaCl/0.0015M sodiumcitrate/0.1% NaDodSO₄ (SDS) at 50° C., or (2) employ duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C. Another example is use of 50%formamide, 5×SSC (0.75M NaCl, 0.075M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/mL), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

"Moderately stringent conditions" are described in Sambrook et al.,supra, and include the use of a washing solution and hybridizationconditions (e.g., temperature, ionic strength, and % SDS) less stringentthan described above. An example of moderately stringent conditions is acondition such as overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37°-50° C. The skilled artisanwill recognize how to adjust the temperature, ionic strength, etc., asnecessary to accommodate factors such as probe length and the like.

"Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

"Native antibodies and immunoglobulins" are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one end (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light- and heavy-chain variable domains (Clothia et al., J.Mol. Biol., 186: 651-663 [1985]; Novotny and Haber, Proc. Natl. Acad.Sci. USA, 82: 4592-4596 [1985]).

The term "variable" refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. [1991]). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called "Fab" fragments, each with a single antigen-bindingsite, and a residual "Fc" fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab')₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

"Fv" is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H) -V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab' fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab'-SH is the designationherein for Fab' in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab')₂ antibody fragments originally wereproduced as pairs of Fab' fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, and IgA-2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term "antibody" is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist and antagonistantibodies) and antibody compositions with polyepitopic specificity.

The term "monoclonal antibody" as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-CHF antibody with a constant domain (e.g. "humanized"antibodies), or a light chain with a heavy chain, or a chain from onespecies with a chain from another species, or fusions with heterologousproteins, regardless of species of origin or immunoglobulin class orsubclass designation, as well as antibody fragments (e.g., Fab, F(ab')₂,and Fv), so long as they exhibit the desired biological activity. [See,e.g. Cabilly, et al., U.S. Pat. No. 4,816,567; Mage & Lamoyi, inMonoclonal Antibody Production Techniques and Applications, pp. 79-97(Marcel Dekker, Inc., New York, 1987).]

Thus, the modifier "monoclonal" indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,Nature, 256: 495 (1975), or may be made by recombinant DNA methods(Cabilly et al., supra).

The monoclonal antibodies herein specifically include "chimeric"antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (Cabilly et al., supra;Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 [1984]).

"Humanized" forms of non-human (e.g., murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab', F(ab')₂, or other antigen-binding subsequencesof antibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary-determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see: Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); andPresta, Curr. Op. Struct. Biol., 2: 593-596 (1992).

"Non-immunogenic in a human" means that upon contacting the polypeptidein a pharmaceutically acceptable carrier and in a therapeuticallyeffective amount with the appropriate tissue of a human, no state ofsensitivity or resistance to the polypeptide is demonstratable upon thesecond administration of the polypeptide after an appropriate latentperiod (e.g., 8 to 14 days).

"Neurological disorder" refers to a disorder of neurons, including bothperipheral neurons and neurons from the central nervous system. Examplesof such disorders include all neurodegenerative diseases, such asperipheral neuropathies (motor and sensory), amyotrophic lateralsclerosis (ALS), Alzheimer's disease, Parkinson's disease, stroke,Huntington's disease, epilepsy, and ophthalmologic diseases such asthose involving the retina, e.g., diabetic retinopathy, retinaldystrophy, and retinal degeneration caused by infantile malignantosteopetrosis, ceroid-lipofuscosis, or cholestasis, or caused byphotodegeneration, trauma, axotomy, neurotoxic-excitatory degeneration,or ischemic neuronal degeneration.

"Peripheral neuropathy" refers to a disorder affecting the peripheralnervous system, most often manifested as one or a combination of motor,sensory, sensorimotor, or autonomic neural dysfunction. The wide varietyof morphologies exhibited by peripheral neuropathies can each beattributed uniquely to an equally wide number of causes. For example,peripheral neuropathies can be genetically acquired, can result from asystemic disease, or can be induced by a toxic agent. Examples includebut are not limited to distal sensorimotor neuropathy, or autonomicneuropathies such as reduced motility of the gastrointestinal tract oratony of the urinary bladder. Examples of neuropathies associated withsystemic disease include post-polio syndrome; examples of hereditaryneuropathies include Charcot-Marie-Tooth disease, Refsum's disease,Abetalipoproteinemia, Tangier disease, Krabbe's disease, Metachromaticleukodystrophy, Fabry's disease, and Dejerine-Sottas syndrome; andexamples of neuropathies caused by a toxic agent include those caused bytreatment with a chemotherapeutic agent such as vincristine.

"Heart failure" refers to an abnormality of cardiac function where theheart does not pump blood at the rate needed for the requirements ofmetabolizing tissues. Heart failure includes a wide range of diseasestates such as congestive heart failure, myocardial infarction, andtachyarrhythmia.

"Treatment" refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein which the disorder is to be prevented.

"Mammal" for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal herein is human.

As used herein, "ACE inhibitor" refers to angiotensin-converting enzymeinhibiting drugs which prevent the conversion of angiotensin I toangiotensin II. The ACE inhibitors may be beneficial in congestive heartfailure by reducing systemic vascular resistance and relievingcirculatory congestion. The ACE inhibitors include but are not limitedto those designated by the trademarks Accupril® (quinapril), Altace®(ramipril), Capoten® (captopril), Lorensin® (benazepril), Monopril®(fosinopril), Prinivil® (lisinopril), Vasotec® (enalapril), and Zestril®(lisinopril). One example of an ACE inhibitor is that sold under thetrademark Capoten®. Generically referred to as captopril, this ACEinhibitor is designated chemically as1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline.

II. Modes for Practicing the Invention 1. CHF Polypeptides

Preferred polypeptides of this invention are substantially homogeneousCHF polypeptide(s), having the biological properties of being myocytehypertrophic and of stimulating the development of chick ciliary neuronsin a CNTF assay. More preferred CHFs are isolated mammalian protein(s)having hypertrophic, anti-arrhythmic, inotropic, and neurologicalactivity, Most preferred polypeptides of this invention are mouse andhuman CHFs including fragments thereof having hypertrophic,anti-arrhythmic, inotropic, and neurological activity. Optionally thesemurine and human CHFs lack glycosylation.

Optional preferred polypeptides of this invention are biologicallyactive CHF variant(s) with an amino acid sequence having at least 70%amino acid sequence identity with the murine CHF of FIG. 1, preferablyat least 75%, more preferably at least 80%, still more preferably atleast 85%, even more preferably at least 90%, and most preferably atleast 95% (i.e., 70-100%, 75-100%, 80-100%, 85-100%, 90-100%, and95-100% sequence identity, respectively). Alternatively, the preferredbiologically active CHF variant(s) have an amino acid sequence having atleast 70%, preferably at least 75%, more preferably at least 80%, stillmore preferably at least 85%, even more preferably at least 90%, andmost preferably at least 95% amino acid sequence identity with the humanCHF sequence of FIG. 5 (i.e., 70-100%, 75-100%, 80-100%, 85-100%,90-100%, and 95-100% sequence identity, respectively).

The CHF cloned from murine embryoid bodies has the followingcharacteristics:

(1) It has a molecular weight of about 21-23 kD as measured by reducingSDS-PAGE;

(2) It shows positive activity in the CNTF chick ciliary neuron assayand in the myocyte hypertrophy and ANP-release hypertrophy assays.

More preferred CHF polypeptides are those encoded by genomic DNA or cDNAand having the amino acid sequence of murine CHF described in FIG. 1 orthe amino acid sequence of human CHF described in FIG. 5.

Other preferred naturally occurring biologically active CHF polypeptidesof this invention include prepro-CHF, pro-CHF, pre-CHF, mature CHF, andglycosylation variants thereof.

Still other preferred polypeptides of this invention include CHFsequence variants and chimeric CHFs. Ordinarily, preferred CHF sequencevariants are biologically active CHF variants that have an amino acidsequence having at least 70% amino acid sequence identity with the humanor murine CHF, preferably at least 75%, more preferably at least 80%,still more preferably at least 85%, even more preferably at least 90%,and most preferably at least 95%. Art exemplary preferred CHF variant isa C-terminal domain CHF variant in which one or more of the basic ordibasic amino acid residue(s) (e.g., R or K) is substituted with anon-basic amino acid residue(s) (e.g., hydrophobic, neutral, acidic,aromatic, gly, pro and the like).

Another exemplary preferred CHF sequence variant is a "domain chimera"that consists of the N-terminal residues substituted with one or more,but not all, of the human CNTF residues approximately aligned as shownin FIG. 2. In this embodiment, the CHF chimera would have individual orblocks of residues from the human CNTF sequence added to or substitutedinto the CHF sequence at positions corresponding to the alignment shownin FIG. 2. For example, one or more of those segments of CNTF that arenot homologous could be substituted into the corresponding segments ofCHF. It is contemplated that this "CHF-CNTF domain chimera" will havemixed hypertrophic/anti-arrhythmic/inotropic/neurotrophic biologicalactivity.

Other preferred polypeptides of this invention include CHF fragmentshaving a consecutive sequence of at least 10, 15, 20, 25, 30, or 40amino acid residues, preferably about 10-150 residues, that is identicalto the sequence of the CHF isolated from murine embryoid bodies or tothat of the corresponding human CHF. Other preferred CHF fragmentsinclude those produced as a result of chemical or enzymatic hydrolysisor digestion of the purified CHF.

Another aspect of the invention is a method for purifying CHF moleculescomprising contacting a CHF source containing the CHF molecules to bepurified with an immobilized receptor or antibody polypeptide, underconditions whereby the CHF molecules to be purified are selectivelyadsorbed onto the immobilized receptor or antibody polypeptide, washingthe immobilized support to remove non-adsorbed material, and eluting themolecules to be purified from the immobilized receptor or antibodypolypeptide to which they are adsorbed with an elution buffer. Thesource containing the CHF may be a cell suspension of embryoid bodies.

Alternatively, the source containing the CHF is recombinant cell culturewhere the concentration of CHF in either the culture medium or in celllysates is generally higher than in plasma or other natural sources. Inthis case the above-described immunoaffinity method, while still useful,is usually not necessary and more traditional protein purificationmethods known in the art may be applied. Briefly, the preferredpurification method to provide substantially homogeneous CHF comprises:removing particulate debris by, for example, centrifugation orultrafiltration; optionally concentrating the protein pool with acommercially available protein concentration filter; and thereafterpurifying the CHF from contaminant soluble proteins and polypeptides,with the following procedures being exemplary of suitable purificationprocedures: by fractionation on immunoaffinity or ion-exchange columns;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; Toyopearl and MONO-Q or MONO-Schromatography; gel filtration using, for example, Sephadex G-75;chromatography on columns that bind the CHF, and protein A Sepharosecolumns to remove contaminants such as IgG. One preferred purificationscheme for both native and recombinant CHF uses a Butyl Toyopearl columnfollowed by a MONO-Q column and a reverse-phase C4 column as describedfurther below.

In another preferred embodiment, this invention provides an isolatedantibody capable of binding to the CHF. A preferred isolated anti-CHFantibody is monoclonal (Kohler and Milstein, Nature, 256: 495-497[1975]; Campbell, Laboratory Techniques in Biochemistry and MolecularBiology, Burdon et al., Eds, Volume 13, Elsevier Science Publishers,Amsterdam [1985]; and Huse et al., Science, 246: 1275-1281 [1989]).Preferred isolated anti-CHF antibody is one that binds to CHF with anaffinity of at least about 10⁶ L/mole. More preferably, the antibodybinds with an affinity of at least about 10⁷ L/mole. Most preferably,the antibody is raised against a CHF having one of the above-describedeffector functions.

The isolated antibody capable of binding to the CHF may optionally befused to a second polypeptide and the antibody or fusion thereof may beused to isolate and purify CHF from a source as described above forimmobilized CHF polypeptide. In a further preferred aspect of thisembodiment, the invention provides a method for detecting the CHF invitro or in vivo comprising contacting the antibody with a sample,especially a serum sample, suspected of containing the CHF and detectingif binding has occurred.

The invention also provides an isolated nucleic acid molecule encodingthe CHF or fragments thereof, which nucleic acid molecule may be labeledor unlabeled with a detectable moiety, and a nucleic acid moleculehaving a sequence that is complementary to, or hybridizes understringent or moderately stringent conditions with, a nucleic acidmolecule having a sequence encoding a CHF. A preferred CHF nucleic acidis RNA or DNA that encodes a biologically active CHF sharing at least75%, more preferably at least 80%, still more preferably at least 85%,even more preferably 90%, and most preferably 95%, sequence identitywith the murine or human CHF.

More preferred isolated nucleic acid molecules are DNA sequencesencoding biologically active CHF, selected from: (a) DNA based on thecoding region of a mammalian CHF gene (e.g., DNA comprising thenucleotide sequence provided in FIG. 1 or FIG. 5, or fragments thereof);(b) DNA capable of hybridizing to a DNA of (a) under at least moderatelystringent conditions; and (c) DNA that is degenerate to a DNA defined in(a) or (b) which results from degeneracy of the genetic code. It iscontemplated that the novel CHFs described herein may be members of afamily of ligands having suitable sequence identity that their DNA mayhybridize with the DNA of FIG. 1 or FIG. 5 (or fragments thereof) underlow to moderate stringency conditions. Thus, a further aspect of thisinvention includes DNA that hybridizes under low to moderate stringencyconditions with DNA encoding the CHF polypeptides.

Preferably, the nucleic acid molecule is cDNA encoding the CHF andfurther comprises a replicable vector in which the cDNA is operablylinked to control sequences recognized by a host transformed with thevector. This aspect further includes host cells transformed with thevector and a method of using the cDNA to effect production of CHF,comprising expressing the cDNA encoding the CHF in a culture of thetransformed host cells and recovering the CHF from the host cellculture. The CHF prepared in this manner is preferably substantiallyhomogeneous murine or human CHF.

The invention further includes a preferred method for treating a mammalhaving heart failure, or an arrhythmic, inotropic, or neurologicaldisorder, comprising administering a therapeutically effective amount ofa CHF to the mammal. Optionally, the CHF is administered in combinationwith an ACE inhibitor, such as captopril, in the case of congestiveheart failure, or with another myocardiotrophic, anti-arrhythmic, orinotropic factor in the case of other types of heart failure or cardiacdisorder, or with a neurotrophic molecule such as, e.g., IGF-I, CNTF,NGF, NT-3, BDNF, NT-4, NT-5, etc. in the case of a neurologicaldisorder.

2. Preparation of Natural-Sequence CHF and Variants

Most of the discussion below pertains to production of CHF by culturingcells transformed with a vector containing CHF nucleic acid andrecovering the polypeptide from the cell culture. It is furtherenvisioned that the CHF of this invention may be produced by homologousrecombination, as provided for in WO 91/06667 published 16 May 1991.Briefly, this method involves transforming primary mammalian cellscontaining endogenous CHF gene (e.g., human cells if the desired CHF ishuman) with a construct (i.e., vector) comprising an amplifiable gene(such as dihydrofolate reductase [DHFR] or others discussed below) andat least one flanking region of a length of at least about 150 bp thatis homologous with a DNA sequence at the locus of the coding region ofthe CHF gene to provide amplification of the CHF gene. The amplifiablegene must be at a site that does not interfere with expression of theCHF gene. The transformation is conducted such that the constructbecomes homologously integrated into the genome of the primary cells todefine an amplifiable region.

Primary cells comprising the construct are then selected for by means ofthe amplifiable gene or other marker present in the construct. Thepresence of the marker gene establishes the presence and integration ofthe construct into the host genome. No further selection of the primarycells need be made, since selection will be made in the second host. Ifdesired, the occurrence of the homologous recombination event can bedetermined by employing PCR and either sequencing the resultingamplified DNA sequences or determining the appropriate length of the PCRfragment when DNA from correct homologous integrants is present andexpanding only those cells containing such fragments. Also if desired,the selected cells may be amplified at this point by stressing the cellswith the appropriate amplifying agent (such as methotrexate if theamplifiable gene is DHFR), so that multiple copies of the target geneare obtained. Preferably, however, the amplification step is notconducted until after the second transformation described below.

After the selection step, DNA portions of the genome, sufficiently largeto include the entire amplifiable region, are isolated from the selectedprimary cells. Secondary mammalian expression host cells are thentransformed with these genomic DNA portions and cloned, and clones areselected that contain the amplifiable region. The amplifiable region isthen amplified by means of an amplifying agent, if not already amplifiedin the primary cells. Finally, the secondary expression host cells nowcomprising multiple copies of the amplifiable region containing CHF aregrown so as to express the gene and produce the protein.

A. Isolation of DNA Encoding CHF

The DNA encoding CHF may be obtained from any cDNA library prepared fromtissue believed to possess the CHF mRNA and to express it at adetectable level. The mRNA is suitably prepared, for example, fromseven-day differentiated embryoid bodies. The CHF gene may also beobtained from a genomic library or by in vitro oligonucleotide synthesisas defined above assuming the complete nucleotide or amino acid sequenceis known.

Libraries are screened with probes designed to identify the gene ofinterest or the protein encoded by it. For cDNA expression libraries,suitable probes include, e.g.: monoclonal or polyclonal antibodies thatrecognize and specifically bind to the CHF; oligonucleotides of about20-80 bases in length that encode known or suspected portions of the CHFcDNA from the same or different species; and/or complementary orhomologous cDNAs or fragments thereof that encode the same or a similargene. Appropriate probes for screening genomic DNA libraries include,but are not limited to, oligonucleotides, cDNAs, or fragments thereofthat encode the same or a similar gene, and/or homologous genomic DNAsor fragments thereof. Screening the cDNA or genomic library with theselected probe may be conducted using standard procedures as describedin chapters 10-12 of Sambrook et al.. supra.

An alternative means to isolate the gene encoding CHF is to use PCRmethodology as described in section 14 of Sambrook et al., supra. Thismethod requires the use of oligonucleotide probes that will hybridize tothe CHF. Strategies for selection of oligonucleotides are describedbelow.

A preferred method of practicing this invention is to use carefullyselected oligonucleotide sequences to screen cDNA libraries from varioustissues, preferably mammalian differentiated embryoid bodies andplacental, cardiac, and brain cell lines. More preferably, humanembryoid, placental, cardiac, and brain cDNA libraries are screened withthe oligonucleotide probes.

The oligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The actual nucleotide sequence(s) is usually based on conserved orhighly homologous nucleotide sequences. The oligonucleotides may bedegenerate at one or more positions. The use of degenerateoligonucleotides may be of particular importance where a library isscreened from a species in which preferential codon usage is not known.

The oligonucleotide must be labeled such that it can be detected uponhybridization to DNA in the library being screened. The preferred methodof labeling is to use ³² P-labeled ATP with polynucleotide kinase, as iswell known in the art, to radiolabel the oligonucleotide. However, othermethods may be used to label the oligonucleotide, including, but notlimited to, biotinylation or enzyme labeling.

Of particular interest is the CHF nucleic acid that encodes afull-length polypeptide. In some preferred embodiments, the nucleic acidsequence includes the native CHF signal sequence. Nucleic acid havingall the protein coding sequence is obtained by screening selected cDNAor genomic libraries using the deduced amino acid sequence disclosedherein for the first time, and, if necessary, using conventional primerextension procedures as described in section 7.79 of Sambrook et al.,supra, to detect precursors and processing intermediates of mRNA thatmay not have been reverse-transcribed into cDNA.

B. Amino Acid Sequence Variants of Native CHF

Amino acid sequence variants of native CHF are prepared by introducingappropriate nucleotide changes into the native CHF DNA, or by in vitrosynthesis of the desired CHF polypeptide. Such variants include, forexample, deletions from, or insertions or substitutions of, residueswithin the amino acid sequence shown for murine CHF in FIG. 1 and forhuman CHF in FIG. 5. Any combination of deletion, insertion, andsubstitution is made to arrive at the final construct, provided that thefinal construct possesses the desired characteristics. Excluded from thescope of this invention are CHF variants or polypeptide sequences thatare the rat homolog of CHF. The amino acid changes also may alterpost-translational processes of the native CHF, such as changing thenumber or position of glycosylation sites.

For the design of amino acid sequence variants of native CHF, thelocation of the mutation site and the nature of the mutation will dependon the CHF characteristic(s) to be modified. For example, candidate CHFantagonists or super agonists will be initially selected by locatingsites that are identical or highly conserved among CHF and other ligandsbinding to members of the growth hormone (GH)/cytokine receptor family,especially CNTF and leukemia inhibitory factor (LIF). The sites formutation can be modified individually or in series, e.g., by (1)substituting first with conservative amino acid choices and then withmore radical selections depending upon the results achieved, (2)deleting the target residue, or (3) inserting residues of the same or adifferent class adjacent to the located site, or combinations of options1-3.

A useful method for identification of certain residues or regions of thenative CHF polypeptide that are preferred locations for mutagenesis iscalled "alanine scanning mutagenesis," as described by Cunningham andWells, Science, 244: 1081-1085 (1989). Here, a residue or group oftarget residues are identified (e.g., charged residues such as arg, asp,his, lys, and glu) and replaced by a neutral or negatively charged aminoacid (most preferably alanine or polyalanine) to affect the interactionof the amino acids with the surrounding aqueous environment in oroutside the cell. Those domains demonstrating functional sensitivity tothe substitutions then are refined by introducing further or othervariants at or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, tooptimize the performance of a mutation at a given site, alanine scanningor random mutagenesis is conducted at the target codon or region and theCHF variants produced are screened for the optimal combination ofdesired activity.

There are two principal variables in the construction of amino acidsequence variants: the location of the mutation site and the nature ofthe mutation. These are variants from the FIG. 1 or FIG. 5 sequence, andmay represent naturally occurring alleles (which will not requiremanipulation of the native CHF DNA) or predetermined mutant forms madeby mutating the DNA, either to arrive at an allele or a variant notfound in nature. In general, the location and nature of the mutationchosen will depend upon the CHF characteristic to be modified.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Contiguous deletions ordinarily are made in even numbers ofresidues, but single or odd numbers of deletions are within the scopehereof. Deletions may be introduced into regions of low homology amongCHF and other ligands binding to the GH/cytokine receptor family whichshare the most sequence identity to the human CHF amino acid sequence tomodify the activity of CHF. Deletions from CHF in areas of substantialhomology with one of the receptor binding sites of other ligands thatbind to the GH/cytokine receptor family will be more likely to modifythe biological activity of CHF more significantly. The number ofconsecutive deletions will be selected so as to preserve the tertiarystructure of CHF in the affected domain, e.g., beta-pleated sheet oralpha helix.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Intrasequence insertions (i.e.,insertions within the mature CHF sequence) may range generally fromabout 1 to 10 residues, more preferably 1 to 5, most preferably 1 to 3.Insertions are preferably made in even numbers of residues, but this isnot required. Examples of terminal insertions include mature CHF with anN-terminal methionyl residue, an artifact of the direct production ofmature CHF in recombinant cell culture, and fusion of a heterologousN-terminal signal sequence to the N-terminus of the mature CHF moleculeto facilitate the secretion of mature CHF from recombinant hosts. Suchsignal sequences generally will be obtained from, and thus homologousto, the intended host cell species. Suitable sequences include STII orlpp for E. coli, alpha factor for yeast, and viral signals such asherpes gD for mammalian cells.

Other insertional variants of the native CHF molecule include the fusionto the N- or C-terminus of native CHF of immunogenic polypeptides, e.g.,bacterial polypeptides such as beta-lactamase or an enzyme encoded bythe E. coli trp locus, or yeast protein, and C-terminal fusions withproteins having a long half-life such as immunoglobulin constant regions(or other immunoglobulin regions), albumin, or ferritin, as described inWO 89/02922 published 6 Apr. 1989.

A third group of variants are amino acid substitution variants. Thesevariants have at least one amino acid residue in the native CHF moleculeremoved and a different residue inserted in its place. The sites ofgreatest interest for substitutional mutagenesis include sitesidentified as the active site(s) of native CHF and sites where the aminoacids found in the known analogues are substantially different in termsof side-chain bulk, charge, or hydrophobicity, but where there is also ahigh degree of sequence identity at the selected site within variousanimal CHF species, or where the amino acids found in known ligands thatbind to members of the GH/cytokine receptor family and novel CHF aresubstantially different in terms of side-chain bulk, charge, orhydrophobicity, but where there also is a high degree of sequenceidentity at the selected site within various animal analogues of suchligands (e.g., among all the animal CNTF molecules). This analysis willhighlight residues that may be involved in the differentiation ofactivity of the cardiac hypertrophic, anti-arrhythmic, inotropic, andneurotrophic factors, and therefore, variations at these sites mayaffect such activities.

Other sites of interest are those in which particular residues of theCHF obtained from various species are identical among all animal speciesof CHF and other ligands binding to GH/cytokine receptor familymolecules, this degree of conformation suggesting importance inachieving biological activity common to these enzymes. These sites,especially those falling within a sequence of at least three otheridentically conserved sites, are substituted in a relativelyconservative manner. Such conservative substitutions are shown in Table1 under the heading of preferred substitutions. If such substitutionsresult in a change in biological activity, then more substantialchanges, denominated exemplary substitutions in Table 1, or as furtherdescribed below in reference to amino acid classes, are introduced andthe products screened.

                  TABLE 1                                                         ______________________________________                                        Original    Exemplary      Preferred                                          Residue     Substitutions  Substitutions                                      ______________________________________                                        Ala    (A)      val; leu; ile  val                                            Arg    (R)      lys; gln; asn  lys                                            Asn    (N)      gln; his; lys; arg                                                                           gln                                            Asp    (D)      glu            glu                                            Cys    (c)      ser            ser                                            Gln    (Q)      asn            asn                                            Glu    (E)      asp            asp                                            Gly    (G)      pro            pro                                            His    (H)      asn; gln; lys; arg                                                                           arg                                            Ile    (I)      leu; val; met; ala; phe;                                                                     leu                                                            norleucine                                                    Leu    (L)      norleucine; ile; val;                                                                        ile                                                            met; ala; phe                                                 Lys    (K)      arg; gln; asn  arg                                            Met    (M)      leu; phe; ile  leu                                            Phe    (F)      leu; val; ile; ala                                                                           leu                                            Pro    (P)      gly            gly                                            Ser    (S)      thr            thr                                            Thr    (T)      ser            ser                                            Trp    (W)      tyr            tyr                                            Tyr    (Y)      trp; phe; thr; ser                                                                           phe                                            Val    (V)      ile; leu; met; phe;                                                                          leu                                                            ala; norleucine                                               ______________________________________                                    

Substantial modifications in function or immunological identity of thenative CHF are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: ash, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

In one embodiment of the invention, it is desirable to inactivate one ormore protease cleavage sites that are present in the molecule. Thesesites are identified by inspection of the encoded amino acid sequence,in the case of trypsin, e.g., for an arginyl or lysinyl residue. Whenprotease cleavage sites are identified, they are rendered inactive toproteolytic cleavage by substituting the targeted residue with anotherresidue, preferably a basic residue such as glutamine or a hydrophobicresidue such as serine; by deleting the residue; or by inserting aprolyl residue immediately after the residue.

In another embodiment, any methionyl residues other than the startingmethionyl residue of the signal sequence, or any residue located withinabout three residues N- or C-terminal to each such methionyl residue, issubstituted by another residue (preferably in accord with Table 1) ordeleted. Alternatively, about 1-3 residues are inserted adjacent to suchsites.

Any cysteine residues not involved in maintaining the properconformation of native CHF also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking.

Nucleic acid molecules encoding amino acid sequence variants of nativeCHF are prepared by a variety of methods known in the art. These methodsinclude, but are not limited to, isolation from a natural source (in thecase of naturally occurring amino acid sequence variants) or preparationby oligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of native CHF.

Oligonucleotide-mediated mutagenesis is a preferred method for preparingsubstitution, deletion, and insertion variants of native CHF DNA. Thistechnique is well known in the art as described by Adelman et al., DNA,2: 183 (1983). Briefly, native CHF DNA is altered by hybridizing anoligonucleotide encoding the desired mutation to a DNA template, wherethe template is the single-stranded form of a plasmid or bacteriophagecontaining the unaltered or native DNA sequence of CHF. Afterhybridization, a DNA polymerase is used to synthesize an entire secondcomplementary strand of the template that will thus incorporate theoligonucleotide primer, and will code for the selected alteration in thenative CHF DNA.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Natl. Acad. Sci. USA, 75: 5765 (1978).

The DNA template can be generated by those vectors that are eitherderived from bacteriophage M13 vectors (the commercially availableM13mp18 and M13mp19 vectors are suitable), or those vectors that containa single-stranded phage origin of replication as described by Viera etal. Meth. Enzymol., 153: 3 (1987). Thus, the DNA that is to be mutatedmay be inserted into one of these vectors to generate single-strandedtemplate. Production of the single-stranded template is described inSections 4.21-4.41 of Sambrook et al., supra.

Alternatively, single-stranded DNA template may be generated bydenaturing double-stranded plasmid (or other) DNA using standardtechniques.

For alteration of the native DNA sequence (to generate amino acidsequence variants, for example), the oligonucleotide is hybridized tothe single-stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually the Klenow fragment of DNA polymeraseI, is then added to synthesize the complementary strand of the templateusing the oligonucleotide as a primer for synthesis. A heteroduplexmolecule is thus formed such that one strand of DNA encodes the mutatedform of native CHF, and the other strand (the original template) encodesthe native, unaltered sequence of CHF. This heteroduplex molecule isthen transformed into a suitable host cell, usually a prokaryote such asE. coli JM101. After the cells are grown, they are plated onto agaroseplates and screened using the oligonucleotide primer radiolabeled with³² P to identify the bacterial colonies that contain the mutated DNA.The mutated region is then removed and placed in an appropriate vectorfor protein production, generally an expression vector of the typetypically employed for transformation of an appropriate host.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthio-deoxyribocytosine called dCTP-(aS) (which can be obtained from theAmersham Corporation). This mixture is added to thetemplate-oligonucleotide complex. Upon addition of DNA polymerase tothis mixture, a strand of DNA identical to the template except for themutated bases is generated. In addition, this new strand of DNA willcontain dCTP-(aS) instead of dCTP, which serves to protect it fromrestriction endonuclease digestion.

After the template strand of the double-stranded heteroduplex is nickedwith an appropriate restriction enzyme, the template strand can bedigested with ExoIII nuclease or another appropriate nuclease past theregion that contains the site(s) to be mutagenized. The reaction is thenstopped to leave a molecule that is only partially single-stranded. Acomplete double-stranded DNA homoduplex is then formed using DNApolymerase in the presence of all four deoxyribonucleotidetriphosphates, ATP, and DNA ligase. This homoduplex molecule can then betransformed into a suitable host cell such as E. coli JM101, asdescribed above.

DNA encoding mutants of native CHF with more than one amino acid to besubstituted may be generated in one of several ways. If the amino acidsare located close together in the polypeptide chain, they may be mutatedsimultaneously using one oligonucleotide that codes for all of thedesired amino acid substitutions. If, however, the amino acids arelocated some distance from each other (separated by more than about tenamino acids), it is more difficult to generate a single oligonucleotidethat encodes all of the desired changes. Instead, one of two alternativemethods may be employed.

In the first method, a separate oligonucleotide is generated for eachamino acid to be substituted. The oligonucleotides are then annealed tothe single-stranded template DNA simultaneously, and the second strandof DNA that is synthesized from the template will encode all of thedesired amino acid substitutions.

The alternative method involves two or more rounds of mutagenesis toproduce the desired mutant. The first round is as described for thesingle mutants: wild-type DNA is used for the template, anoligonucleotide encoding the first desired amino acid substitution(s) isannealed to this template, and the heteroduplex DNA molecule is thengenerated. The second round of mutagenesis utilizes the mutated DNAproduced in the first round of mutagenesis as the template. Thus, thistemplate already contains one or more mutations. The oligonucleotideencoding the additional desired amino acid substitution(s) is thenannealed to this template, and the resulting strand of DNA now encodesmutations from both the first and second rounds of mutagenesis. Thisresultant DNA can be used as a template in a third round of mutagenesis,and so on.

PCR mutagenesis is also suitable for making amino acid variants ofnative CHF. While the following discussion refers to DNA, it isunderstood that the technique also finds application with RNA. The PCRtechnique generally refers to the following procedure (see Erlich,supra, the chapter by R. Higuchi, p. 61-70): When small amounts oftemplate DNA are used as starting material in a PCR, primers that differslightly in sequence from the corresponding region in a template DNA canbe used to generate relatively large quantities of a specific DNAfragment that differs from the template sequence only at the positionswhere the primers differ from the template. For introduction of amutation into a plasmid DNA, one of the primers is designed to overlapthe position of the mutation and to contain the mutation; the sequenceof the other primer must be identical to a stretch of sequence of theopposite strand of the plasmid, but this sequence can be locatedanywhere along the plasmid DNA. It is preferred, however, that thesequence of the second primer is located within 200 nucleotides fromthat of the first, such that in the end the entire amplified region ofDNA bounded by the primers can be easily sequenced. PCR amplificationusing a primer pair like the one just described results in a populationof DNA fragments that differ at the position of the mutation specifiedby the primer, and possibly at other positions, as template copying issomewhat error-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer, or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more)-partligation.

In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) islinearized by digestion with a restriction endonuclease that has aunique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide triphosphates and isincluded in the GeneAmp® kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 μL. The reaction mixtureis overlayed with 35 μL mineral oil. The reaction mixture is denaturedfor five minutes at 100° C., placed briefly on ice, and then 1 μLThermus aquaticus (Taq) DNA polymerase (5 units/μL, purchased fromPerkin-Elmer Cetus) is added below the mineral oil layer. The reactionmixture is then inserted into a DNA Thermal Cycler (purchased fromPerkin-Elmer Cetus) programmed as follows:

2 min. 55° C.

30 sec. 72° C., then 19 cycles of the following:

30 sec. 94° C.

30 sec. 55° C., and

30 sec. 72° C.

At the end of the program, the reaction vial is removed from the thermalcycler and the aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to the appropriate treatments for insertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al., Gene, 34: 315 (1985). Thestarting material is the plasmid (or other vector) comprising the nativeCHF DNA to be mutated. The codon(s) in the native CHF DNA to be mutatedare identified. There must be a unique restriction endonuclease site oneach side of the identified mutation site(s). If no such restrictionsites exist, they may be generated using the above-describedoligonucleotide-mediated mutagenesis method to introduce them atappropriate locations in the native CHF DNA. After the restriction siteshave been introduced into the plasmid, the plasmid is cut at these sitesto linearize it. A double-stranded oligonucleotide encoding the sequenceof the DNA between the restriction sites but containing the desiredmutation(s) is synthesized using standard procedures. The two strandsare synthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 3' and 5' ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the CHF DNAsequence mutated from native CHF.

C. Insertion of Nucleic Acid into Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding CHF is insertedinto a replicable vector for further cloning (amplification of the DNA)or for expression. Many vectors are available, and selection of theappropriate vector will depend on 1) whether it is to be used for DNAamplification or for DNA expression, 2) the size of the nucleic acid tobe inserted into the vector, and 3) the host cell to be transformed withthe vector. Each vector contains various components depending on itsfunction (amplification of DNA or expression of DNA) and the host cellwith which it is compatible. The vector components generally include,but are not limited to, one or more of the following: a signal sequence,an origin of replication, one or more marker genes, an enhancer element,a promoter, and a transcription termination sequence.

(i) Signal Sequence Component

The CHFs of this invention may be produced not only directly, but alsoas a fusion with a heterologous polypeptide, preferably a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide. In general, the signalsequence may be a component of the vector, or it may be a part of theCHF DNA that is inserted into the vector. The heterologous signalsequence selected should be one that is recognized and processed (i.e.,cleaved by a signal peptidase) by the host cell. For prokaryotic hostcells that do not recognize and process the native CHF signal sequence,the signal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group consisting of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.For yeast secretion the native signal sequence may be substituted by,e.g., the yeast invertase leader, yeast alpha factor leader (includingSaccharomyces and Kluyveromyces α-factor leaders, the latter describedin U.S. Pat. No. 5,010,182 issued 23 Apr. 1991), yeast acid phosphataseleader, mouse salivary amylase leader, carboxypeptidase leader, yeastBAR1 leader, Humicola lanuginosa lipase leader, the C. albicansglucoamylase leader (EP 362,179 published 4 Apr. 1990), or the signaldescribed in WO 90/13646 published 15 Nov. 1990. In mammalian cellexpression the native human signal sequence (i.e., the CHF presequencethat normally directs secretion of native CHF from human cells in vivo)is satisfactory, although other mammalian signal sequences may besuitable, such as signal sequences from other animal CHFs, signalsequences from a ligand binding to another GH/cytokine receptor familymember, and signal sequences from secreted polypeptides of the same orrelated species, as well as viral secretory leaders, for example, theherpes simplex gD signal.

The DNA for such precursor region is ligated in reading frame to DNAencoding the mature CHF.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV, orBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Most expression vectors are "shuttle" vectors, i.e., they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of CHF DNA. However, the recovery of genomic DNA encoding CHFis more complex than that of an exogenously replicated vector becauserestriction enzyme digestion is required to excise the CHF DNA.

(iii) Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin (Southern et al., J. Molec. Appl. Genet., 1: 327[1982]), mycophenolic acid (Mulligan et al., Science, 209: 1422 [1980]),or hygromycin (Sugden et al., Mol. Cell. Biol., 5: 410-413 [1985]). Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theCHF nucleic acid, such as DHFR or thymidine kinase. The mammalian celltransformants are placed under selection pressure that only thetransformants are uniquely adapted to survive by virtue of having takenup the marker. Selection pressure is imposed by culturing thetransformants under conditions in which the concentration of selectionagent in the medium is successively changed, thereby leading toamplification of both the selection gene and the DNA that encodes CHF.Amplification is the process by which genes in greater demand for theproduction of a protein critical for growth are reiterated in tandemwithin the chromosomes of successive generations of recombinant cells.Increased quantities of CHF are synthesized from the amplified DNA.Other examples of amplifiable genes include metallothionein-I and -II,preferably primate metallothionein genes, adenosine deaminase, ornithinedecarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216 (1980). The transformed cells are then exposed toincreased levels of methotrexate. This leads to the synthesis ofmultiple copies of the DHFR gene, and, concomitantly, multiple copies ofother DNA comprising the expression vectors, such as the DNA encodingCHF. This amplification technique can be used with any otherwisesuitable host, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presenceof endogenous DHFR if, for example, a mutant DHFR gene that is highlyresistant to Mtx is employed (EP 117,060).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding CHF, wild-type DHFR protein, and another selectable marker suchas aminoglycoside 3-phosphotransferase (APH) can be selected by cellgrowth in medium containing a selection agent for the selectable markersuch as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, orG418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 [1979];Kingsman et al., Gene, 7: 141 [1979]; or Tschemper et al., Gene, 10: 157[1980]). The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 (Jones, Genetics, 85: 12 [1977]). The presence ofthe trp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Bianchi et al.,Curr. Genet., 12: 185 (1987). More recently, an expression system forlarge-scale production of recombinant calf chymosin was reported for K.lactis. Van den Berg, Bio/Technology, 8: 135 (1990). Stable multi-copyexpression vectors for secretion of mature recombinant human serumalbumin by industrial strains of Kluyveromyces have also been disclosed.Fleer et al., Bio/Technology, 9: 968-975 (1991).

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the CHFnucleic acid. Promoters are untranslated sequences located upstream (5')to the start codon of a structural gene (generally within about 100 to1000 bp) that control the transcription and translation of particularnucleic acid sequence, such as the CHF nucleic acid sequence, to whichthey are operably linked. Such promoters typically fall into twoclasses, inducible and constitutive. Inducible promoters are promotersthat initiate increased levels of transcription from DNA under theircontrol in response to some change in culture conditions, e.g., thepresence or absence of a nutrient or a change in temperature. At thistime a large number of promoters recognized by a variety of potentialhost cells are well known. These promoters are operably linked toCHF-encoding DNA by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the isolated promotersequence into the vector. Both the native CHF promoter sequence and manyheterologous promoters may be used to direct amplification and/orexpression of the CHF DNA. However, heterologous promoters arepreferred, as they generally permit greater transcription and higheryields of recombinantly produced CHF as compared to the native CHFpromoter.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature, 275: 615[1978]; and Goeddel et al., Nature, 281: 544 [1979]), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8: 4057 [1980] and EP 36,776), and hybrid promoters such as thetac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25[1983]). However, other known bacterial promoters are suitable. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to DNA encoding CHF (Siebenlist et al.,Cell, 20: 269 [1980]) using linkers or adaptors to supply any requiredrestriction sites. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding CHF.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3' end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3' end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem., 255: 2073 [1980]) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg., 7: 149 [1968]; and Holland, Biochemistry, 17: 4900[1978]), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657. Yeast enhancers also are advantageouslyused with yeast promoters.

CHF transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, arian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter, fromheat-shock promoters, and from the promoter normally associated with theCHF sequence, provided such promoters are compatible with the host cellsystems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., Nature, 273:113 (1978); Mulligan and Berg,Science, 209: 1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci.USA, 78: 7398-7402 (1981). The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., Gene, 18: 355-360 (1982). A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See also Gray et al.,Nature, 295: 503-508 (1982) on expressing cDNA encoding immuneinterferon in monkey cells; Reyes et al., Nature, 297: 598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus; Canaani andBerg, Proc. Natl. Acad. Sci. USA, 79: 5166-5170 (1982) on expression ofthe human interferon β1 gene in cultured mouse and rabbit cells; andGorman et al., Proc. Natl. Acad. Sci. USA, 79: 6777-6781 (1982) onexpression of bacterial CAT sequences in CV-1 monkey kidney cells,chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, andmouse NIH-3T3 cells using the Rous sarcoma virus long terminal repeat asa promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the CHF of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent, havingbeen found 5' (Laimins et al., Proc. Natl. Acad. Sci. USA, 78: 993[1981]) and 3' (Lusky et al., Mol. Cell Bio., 3: 1108 [1983]) to thetranscription unit, within an intron (Banerji et al., Cell, 33: 729[1983]), as well as within the coding sequence itself (Osborne et al.,Mol. Cell Bio., 4: 1293 [1984]). Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature, 297: 17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5' or 3' to theCHF-encoding sequence, but is preferably located at a site 5' from thepromoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5' and, occasionally 3', untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding CHF.

(vii) Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9: 309 (1981) or by the method of Maxam et al., Methods inEnzymology, 65: 499 (1980).

(viii) Transient Expression Vectors

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding CHF. In general, transient expression involves the use ofan expression vector that is able to replicate efficiently in a hostcell, such that the host cell accumulates many copies of the expressionvector and, in turn, synthesizes high levels of a desired polypeptideencoded by the expression vector. Sambrook et al., supra, pp.16.17-16.22. Transient expression systems, comprising a suitableexpression vector and a host cell, allow for the convenient positiveidentification of polypeptides encoded by cloned DNAs, as well as forthe rapid screening of such polypeptides for desired biological orphysiological properties. Thus, transient expression systems areparticularly useful in the invention for purposes of identifying analogsand variants of native CHF that are biologically active CHF.

(ix) Suitable Exemplary Vertebrate Cell Vectors

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of CHF in recombinant vertebrate cell culture are described inGething et al., Nature, 293: 620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058. A particularly useful plasmidfor mammalian cell culture production of CHF is pRK5 (EP 307,247) orpSVI6B (WO 91/08291 published 13 Jun. 1991). The pRK5 derivative pRK5B(Holmes et al., Science, 253: 1278-1280 [1991]) is particularly suitableherein for such expression.

D. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast, or higher eukaryote cells described above. Suitableprokaryotes for this purpose include eubacteria, such as Gram-negativeor Gram-positive organisms, for example, Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis and B.licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, andStreptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), E. coli DH5α, and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting. Strain W3110 isone particularly preferred host or parent host because it is a commonhost strain for recombinant DNA product fermentations. Preferably, thehost cell secretes minimal amounts of proteolytic enzymes. For example,strain W3110 may be modified to effect a genetic mutation in the genesencoding proteins endogenous to the host, with examples of such hostsincluding E. coli W3110 strain 1A2, which has the complete genotypetonAΔ; E. coli W3110 strain 9E4, which has the complete genotype tonAΔptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the completegenotype tonA ptr3 phoAΔE15 Δ(argF-lac) 169 ΔdegP ΔompT kan^(r) ; E.coli W3110 strain 37D6, which has the complete genotype tonA ptr3phoAΔE15 Δ(argF-lac) 169 ΔdegP ΔompT Δrbs7 ilvG kan^(r) ; E. coli W3110strain 40B4, which is strain 37D6 with a non-kanamycin resistant degPdeletion mutation; and an E. coli strain having mutant periplasmicprotease disclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990.Alternatively, in vitro methods of cloning, e.g., PCR or other nucleicacid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for CHF-encodingvectors. Saccharomyces cerevisiae, or common baker's yeast, is the mostcommonly used among lower eukaryotic host microorganisms. However, anumber of other genera, species, and strains are commonly available anduseful herein, such as Schizosaccharomyces pombe (Beach and Nurse,Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyceshosts (U.S. Pat. No. 4,943,529; Fleer et al., supra) such as, e.g., K.lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 737[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., supra), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070;Sreekrishna et al., J. Basic Microbiol., 28: 265-278 [1988]); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc.Natl. Acad. Sci. USA, 76: 5259-5263 [1979]); Schwanniomyces such asSchwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A.nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289[1983]; Tilburn et al., Gene, 26: 205-221 [1983]; Yelton et al., Proc.Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and A. niger (Kelly andHynes, EMBO J., 4: 475-479 [1985]).

Suitable host cells for the production of CHF are derived frommulticellular organisms. Such host cells are capable of complexprocessing and glycosylation activities. In principle, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. See, e.g., Luckow et al., Bio/Technology, 6: 47-55(1988); Miller et al., in Genetic Engineering, Setlow, J. K. et al.,eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al.,Nature, 315: 592-594 (1985). A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the CHF DNA. During incubation of the plant cell culture with A.tumefaciens, the DNA encoding the CHF is transferred to the plant cellhost such that it is transfected, and will, under appropriateconditions, express the CHF DNA. In addition, regulatory and signalsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.Depicker et al., J. Mol. Appl. Gen., 1: 561 (1982). In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. EP321,196 published 21 Jun. 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure in recent years (Tissue Culture, Academic Press, Kruse andPatterson, editors [1973]). Examples of useful mammalian host cell linesare a monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL1651); a human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36: 59[1977]); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77: 4216 [1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 [1980]); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumorcells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y.Acad. Sci., 383: 44-68 [1982]); MRC 5 cells; FS4 cells; and a humanhepatoma line (Hep G2).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., supra, or electroporation is generally used for prokaryotes orother cells that contain substantial cell-wall barriers. Infection withAgrobacterium tumefaciens is used for transformation of certain plantcells, as described by Shaw et al., Gene, 23: 315 (1983) and WO 89/05859published 29 Jun. 1989. In addition, plants may be transfected usingultrasound treatment as described in WO 91/00358 published 10 Jan. 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52: 456-457(1978) is preferred. General aspects of mammalian cell host systemtransformations have been described by Axel in U.S. Pat. No. 4,399,216issued 16 Aug. 1983. Transformations into yeast are typically carriedout according to the method of Van Solingen et al., J. Bact., 130: 946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979).However, other methods for introducing DNA into cells, such as bynuclear microinjection, electroporation, bacterial protoplast fusionwith intact cells, or polycations, e.g., polybrene, polyornithine, etc.,may also be used. For various techniques for transforming mammaliancells, see Keown et al., Methods in Enzymology, 185: 527-537 (1990) andMansour et al., Nature, 336: 348-352 (1988).

E. Culturing the Host Cells

Prokaryotic cells used to produce the CHF polypeptide of this inventionare cultured in suitable media as described generally in Sambrook etal., supra.

The mammalian host cells used to produce the CHF of this invention maybe cultured in a variety of media. Commercially available media such asHam's F-10 (Sigma), F-12 (Sigma), Minimal Essential Medium ([MEM],Sigma), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium ([D-MEM],Sigma), and D-MEM/F-12 (Gibco BRL) are suitable for culturing the hostcells. In addition, any of the media described, for example, in Ham andWallace, Methods in Enzymology, 58: 44 (1979); Barnes and Sato, Anal.Biochem., 102: 255 (1980); U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 5,122,469; or 4,560,655; U.S. Pat. Re. No. 30,985; WO90/03430; or WO 87/00195 may be used as culture media for the hostcells. Any of these media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, aprotinin,and/or epidermal growth factor [EGF]), salts (such as sodium chloride,calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides(such as adenosine and thymidine), antibiotics (such as Gentamycin™drug), trace elements (defined as inorganic compounds usually present atfinal concentrations in the micromolar range), and glucose or anequivalent energy source. Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH, andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of in vitro mammalian cell cultures can befound in Mammalian Cell Biotechnology: a Practical Approach, M. Butler,ed. (IRL Press, 1991).

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

F. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77: 5201-5205 [1980]), dot blotting (DNA analysis), orin situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Various labels may be employed, mostcommonly radioisotopes, particularly ³² P. However, other techniques mayalso be employed, such as using biotin-modified nucleotides forintroduction into a polynucleotide. The biotin then serves as the sitefor binding to avidin or antibodies, which may be labeled with a widevariety of labels, such as radionuclides, fluorescers, enzymes, or thelike. Alternatively, antibodies may be employed that can recognizespecific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNAhybrid duplexes or DNA-protein duplexes. The antibodies in turn may belabeled and the assay may be carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.,75: 734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native CHF polypeptide or against a synthetic peptide based onthe DNA sequences provided herein as described further in Section 4below.

G. Purification of CHF Polypeptide

CHF preferably is recovered from the culture medium as a secretedpolypeptide, although it also may be recovered from host cell lysateswhen directly produced without a secretory signal.

When CHF is produced in a recombinant cell other than one of humanorigin, the CHF is completely free of proteins or polypeptides of humanorigin. However, it is necessary to purify CHF from cell proteins orpolypeptides to obtain preparations that are substantially homogeneousas to CHF. As a first step, the particulate debris, either host cells orlysed fragments, is removed, for example, by centrifugation orultrafiltration; optionally, the protein may be concentrated with acommercially available protein concentration filter, followed byseparating the CHF from other impurities by one or more steps selectedfrom immunoaffinity chromatography, ion-exchange column fractionation(e.g., on DEAE or matrices containing carboxymethyl or sulfopropylgroups), chromatography on Blue-Sepharose, CM Blue-Sepharose, MONO-Q,MONO-S, lentil lectin-Sepharose, WGA-Sepharose, Con A-Sepharose, EtherToyopearl, Butyl Toyopearl, Phenyl Toyopearl, or protein A Sepharose,SDS-PAGE chromatography, silica chromatography, chromatofocusing,reverse phase HPLC (e.g., silica gel with appended aliphatic groups),gel filtration using, e.g., Sephadex molecular sieve or size-exclusionchromatography, chromatography on columns that selectively bind the CHF,and ethanol or ammonium sulfate precipitation. A protease inhibitor maybe included in any of the foregoing steps to inhibit proteolysis.Examples of suitable protease inhibitors include phenylmethylsulfonylfluoride (PMSF), leupeptin, pepstatin, aprotinin,4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride-bestatin,chymostatin, and benzamidine.

A preferred purification scheme involves adjusting the culture mediumconditioned by cells transfected with the relevant clone to 1.5M NaCland applying to a Butyl Toyopearl™ column. The column is washed withTris[hydroxymethyl]aminomethane hydrochloride (TRIS-HCl), pH 7.5,containing NaCl, and the activity eluted with TRIS-HCl, pH 7.5,containing 10 mM Zwittergent™ 3-10 surfactant. The peak of activity isadjusted to 150 mM NaCl, pH 8.0, and applied to a MONO-Q Fast Flowcolumn. This column is washed with TRIS-HCl, pH 8.0, containing NaCl andoctyl glucoside. Activity is found in the flow-through fraction. Theactive material is then applied to a reverse phase C4 column in 0.1%TFA, 10% acetonitrile, and eluted with a gradient of 0.1% TFA up to 80%.The activity fractionates at about 15-30 kDa on gel filtration columns.It is expected that a chaotrope such as guanidine-HCl is required forresolution and recovery.

CHF variants in which residues have been deleted, inserted, orsubstituted are recovered in the same fashion as native CHF, takingaccount of any substantial changes in properties occasioned by thevariation. For example, preparation of a CHF fusion with another proteinor polypeptide, e.g., a bacterial or viral antigen, facilitatespurification; an immunoaffinity column containing antibody to theantigen can be used to adsorb the fusion polypeptide. Immunoaffinitycolumns such as a rabbit polyclonal anti-CHF column can be employed toabsorb the CHF variant by binding it to at least one remaining immuneepitope. A protease inhibitor such as those defined above also may beuseful to inhibit proteolytic degradation during purification, andantibiotics may be included to prevent the growth of adventitiouscontaminants. One skilled in the art will appreciate that purificationmethods suitable for native CHF may require modification to account forchanges in the character of CHF or its variants upon production inrecombinant cell culture.

H. Covalent Modifications of CHF Polypeptides

Covalent modifications of CHF polypeptides are included within the scopeof this invention. Both native CHF and amino acid sequence variants ofnative CHF may be covalently modified. One type of covalent modificationincluded within the scope of this invention is the preparation of avariant CHF fragment. Variant CHF fragments having up to about 40 aminoacid residues may be conveniently prepared by chemical synthesis or byenzymatic or chemical cleavage of the full-length or variant CHFpolypeptide. Other types of covalent modifications of the CHF orfragments thereof are introduced into the molecule by reacting targetedamino acid residues of the CHF or fragments thereof with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2- chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵ I or ¹³¹ I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R--N═C═N--R'), where R and R' are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking CHFto a water-insoluble support matrix or surface for use in the method forpurifying anti-CHF antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the CHF polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. By altering is meant deletingone or more carbohydrate moieties found in native CHF, and/or adding oneor more glycosylation sites that are not present in the native CHF.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the CHF polypeptide is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thenative CHF sequence (for O-linked glycosylation sites). For ease, thenative CHF amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the native CHFpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids. The DNA mutation(s) may bemade using methods described above under Section 2B.

Another means of increasing the number of carbohydrate moieties on theCHF polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. These procedures are advantageous in that they do notrequire production of the polypeptide in a host cell that hasglycosylation capabilities for N- or O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published 11 Sep.1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306(1981).

Removal of any carbohydrate moieties present on the CHF polypeptide maybe accomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddin,et al., Arch. Biochem. Biophys., 259: 52 (1987) and by Edge et al.,Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydratemoieties on polypeptides can be achieved by the use of a variety ofendo- and exo-glycosidases as described by Thotakura et al., Meth.Enzymol., 138: 350 (1987).

Glycosylation at potential glycosylation sites may be prevented by theuse of the compound tunicamycin as described by Duskin et al., J. Biol.Chem., 257: 3105 (1982). Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of CHF comprises linking the CHFpolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

CHF also may be entrapped in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization (for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-[methylmethacylate] microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980).

CHF preparations are also useful in generating antibodies, as standardsin assays for CHF (e.g., by labeling CHF for use as a standard in aradioimmunoassay, enzyme-linked immunoassay, or radioreceptor assay), inaffinity purification techniques, and in competitive-type receptorbinding assays when labeled with radioiodine, enzymes, fluorophores,spin labels, and the like.

Since it is often difficult to predict in advance the characteristics ofa variant CHF, it will be appreciated that some screening of therecovered variant will be needed to select the optimal variant. One canscreen for enhanced cardiac hypertrophic, anti-arrhythmic, inotropic, orneurotrophic activity, possession of CHF antagonist activity, increasedexpression levels, oxidative stability, ability to be secreted inelevated yields, and the like. For example, a change in theimmunological character of the CHF molecule, such as affinity for agiven antibody, is measured by a competitive-type immunoassay. Thevariant is assayed for changes in the suppression or enhancement of itshypertrophic, anti-arrhythmic, inotropic, and neurotrophic activities bycomparison to the respective activities observed for native CHF in thesame assay (using, for example, the hypertrophy and neurotrophic assaysdescribed in the examples below.) Other potential modifications ofprotein or polypeptide properties such as redox or thermal stability,hydrophobicity, susceptibility to proteolytic degradation, or thetendency to aggregate with carriers or into multimers are assayed bymethods well known in the art.

I. Antagonists of CHF

Antagonists to CHF can be prepared by using the predicted family ofreceptors for CHF (the GH/cytokine receptor family, including the CNTF,LIF, and oncostatin M receptor subfamily). Thus, the receptor can beexpression cloned from the family; then a soluble form of the receptoris made by identifying the extracellular domain and excising thetransmembrane domain therefrom. The soluble form of the receptor canthen be used as an antagonist, or the receptor can be used to screen forsmall molecules that would antagonize CHF activity.

Alternatively, using the murine sequence shown in FIG. 1 or the humansequence shown in FIG. 5, variants of native CHF are made that act asantagonists. Since the GH/cytokine receptor family is known to have twobinding sites on the ligand, the receptor binding sites of CHF can bedetermined by binding studies and one of them eliminated by standardtechniques (deletion or radical substitution) so that the molecule actsas an antagonist.

Antagonist activity can be determined by several means, including thehypertrophy and neurotrophic assays described herein.

J. Hypertrophy Assay

A miniatured assay is preferably used to assay for hypertrophicactivity. In this assay the medium used allows the cells to survive at alow plating density without serum. By plating directly into this medium,washing steps are eliminated so that fewer cells are removed. Theplating density is important: many fewer cells and the survival isreduced; many more cells and the myocytes begin to self-inducehypertrophy.

The steps involved are:

(a) plating 96-well plates with a suspension of myocytes at a celldensity of about 7.5×10⁴ cells per mL in D-MEM/F-12 medium supplementedwith at least insulin, transferrin, and aprotinin;

(b) culturing the cells;

(c) adding a substance to be assayed (such as one suspected ofcontaining a CHF);

(d) culturing the cells with the substance; and

(e) measuring for hypertrophy.

The medium can be supplemented with additional elements such as EGF thatensure a longer viability of the cells, but such supplements are notessential. D-MEM/F-12 medium is available from Gibco BRL, Gaithersburg,Md., and consists of one of the following media:

    __________________________________________________________________________    11320      11321                                                                              11330                                                                              11331                                                    1 ×  1 ×                                                                          1 ×                                                                          1 ×                                                                          12400                                                                              12500                                          Liquid     Liquid                                                                             Liquid                                                                             Liquid                                                                             Powder                                                                             Powder                                         (mg/L)     (mg/L)                                                                             (mg/L)                                                                             (mg/L)                                                                             (mg/L)                                                                             (mg/L)                                         __________________________________________________________________________    AMINO                                                                         ACIDS:                                                                        L-Ala-                                                                              4.45 4.45 4.45 4.45 4.45 4.45                                           nine                                                                          L-Arg-                                                                              147.50                                                                             147.50                                                                             147.50                                                                             147.50                                                                             147.50                                                                             147.50                                         inine                                                                         .HCl                                                                          L-Asp-                                                                              7.50 7.50 7.50 7.50 7.50 7.50                                           ara-                                                                          gine                                                                          .H.sub.2 O                                                                    L-Asp-                                                                              6.65 6.65 6.65 6.65 6.65 6.65                                           artic                                                                         acid                                                                          L-Cys-                                                                              17.56                                                                              17.56                                                                              17.56                                                                              17.56                                                                              17.56                                                                              17.56                                          teine                                                                         .HCl                                                                          .H.sub.2 O                                                                    L-Cys-                                                                              31.29                                                                              31.29                                                                              31.29                                                                              31.29                                                                              31.29                                                                              31.29                                          tine                                                                          .2HCl                                                                         L-Glu-                                                                              7.35 7.35 7.35 7.35 7.35 7.35                                           tamic                                                                         acid                                                                          L-Glu-                                                                              365.00                                                                             365.00                                                                             365.00                                                                             365.00                                                                             365.00                                                                             365.00                                         tamine                                                                        Gly-  18.75                                                                              18.75                                                                              18.75                                                                              18.75                                                                              18.75                                                                              18.75                                          cine                                                                          L-His-                                                                              31.48                                                                              31.48                                                                              31.48                                                                              31.48                                                                              31.48                                                                              31.48                                          tidine                                                                        .HCl                                                                          .H.sub.2 O                                                                    L-Iso-                                                                              54.47                                                                              54.47                                                                              54.47                                                                              54.47                                                                              54.47                                                                              54.47                                          leu-                                                                          cine                                                                          L-Leu-                                                                              59.05                                                                              59.05                                                                              59.05                                                                              59.05                                                                              59.05                                                                              59.05                                          cine                                                                          L-Lys-                                                                              91.25                                                                              91.25                                                                              91.25                                                                              91.25                                                                              91.25                                                                              91.25                                          ine                                                                           .HCl                                                                          L-    17.24                                                                              17.24                                                                              17.24                                                                              17.24                                                                              17.24                                                                              17.24                                          Meth-                                                                         ionine                                                                        L-    35.48                                                                              35.48                                                                              35.48                                                                              35.48                                                                              35.48                                                                              35.48                                          Phen-                                                                         ylala-                                                                        nine                                                                          L-Pro-                                                                              17.25                                                                              17.25                                                                              17.25                                                                              17.25                                                                              17.25                                                                              17.25                                          line                                                                          L-Ser-                                                                              26.25                                                                              26.25                                                                              26.25                                                                              26.25                                                                              26.25                                                                              26.25                                          ine                                                                           L-    53.45                                                                              53.45                                                                              53.45                                                                              53.4S                                                                              53.45                                                                              53.45                                          Thre-                                                                         onine                                                                         L-    9.02 9.02 9.02 9.02 9.02 9.02                                           Tryp-                                                                         tophan                                                                        L-    55.79                                                                              55.79                                                                              55.79                                                                              55.79                                                                              55.79                                                                              55.79                                          Tyro-                                                                         sine                                                                          .2Na                                                                          .2H.sub.2 O                                                                   L-Val-                                                                              52.85                                                                              52.85                                                                              52.85                                                                              52.85                                                                              52.85                                                                              52.85                                          ine                                                                           INOR-                                                                         GANIC                                                                         SALTS:                                                                        CaCl.sub.2                                                                          116.60                                                                             116.60                                                                             116.60                                                                             116.60                                                                             116.60                                                                             116.60                                         anhyd.                                                                        CUSO.sub.4                                                                          0.0013                                                                             0.0013                                                                             0.0013                                                                             0.0013                                                                             0.0013                                                                             0.0013                                         .5H.sub.2 O                                                                   Fe    0.05 0.05 0.05 0.05 0.05 0.05                                           (NO.sub.3).sub.3                                                              .9H.sub.2 O                                                                   FeSO.sub.4                                                                          0.417                                                                              0.417                                                                              0.417                                                                              0.417                                                                              0.417                                                                              0.417                                          .7H.sub.2 O                                                                   KCl   311.80                                                                             311.80                                                                             311.80                                                                             311.80                                                                             311.80                                                                             311.80                                         MgCl.sub.2                                                                          28.64                                                                              28.64                                                                              28.64                                                                              28.64                                                                              28.64                                                                              28.64                                          MgSO.sub.4                                                                          48.84                                                                              48.84                                                                              48.84                                                                              48.84                                                                              48.84                                                                              48.84                                          NaCl  6999.50                                                                            6999.50                                                                            6999.50                                                                            6999.50                                                                            6999.50                                                                            6999.50                                        NaHCO.sub.3                                                                         2438.00                                                                            2438.00                                                                            2438.00                                                                            2438.00                                                                            --   --                                             NaH.sub.2 PO.sub.4                                                                  62.50                                                                              62.50                                                                              62.50                                                                              --   62.50                                                                              62.50                                          .H.sub.2 O                                                                    Na.sub.2 HPO.sub.4                                                                  71.02                                                                              71.02                                                                              71.02                                                                              --   71.02                                                                              71.02                                          ZnSO.sub.4                                                                          0.432                                                                              0.432                                                                              0.432                                                                              0.432                                                                              0.432                                                                              0.432                                          .7H.sub.2 O                                                                   OTHER                                                                         COMPO-                                                                        NENTS:                                                                        D-Glu-                                                                              3151.00                                                                            3151.00                                                                            3151.00                                                                            3151.00                                                                            3151.00                                                                            3151.00                                        cose                                                                          HEPES --   --   3574.50                                                                            3574.50                                                                            3574.50                                                                            --                                             Na    2.39 2.39 2.39 2.39 2.39 2.39                                           hypo-                                                                         xan-                                                                          thine                                                                         Lino- 0.042                                                                              0.042                                                                              0.042                                                                              0.042                                                                              0.042                                                                              0.042                                          leic                                                                          acid                                                                          Lipoic                                                                              0.105                                                                              0.105                                                                              0.105                                                                              0.105                                                                              0.105                                                                              0.105                                          acid                                                                          Phenol                                                                              8.10 8.10 8.10 8.10 8.10 8.10                                           red                                                                           Pu-   0.081                                                                              0.081                                                                              0.081                                                                              0.081                                                                              0.081                                                                              0.081                                          tres-                                                                         cine                                                                          .2H.sub.2 O                                                                   Sodium                                                                              55.00                                                                              55.00                                                                              55.00                                                                              55.00                                                                              55.00                                                                              55.00                                          pyru-                                                                         vate                                                                          VITA-                                                                         MINS:                                                                         Biotin                                                                              0.0035                                                                             0.0035                                                                             0.0035                                                                             0.0035                                                                             0.0035                                                                             0.0035                                         D-Ca  2.24 2.24 2.24 2.24 2.24 2.24                                           panto-                                                                        then-                                                                         ate                                                                           Cho-  8.98 8.98 8.98 8.98 8.98 8.98                                           line                                                                          chlor-                                                                        ide                                                                           Folic 2.65 2.65 2.65 2.65 2.65 2.65                                           acid                                                                          i-Ino-                                                                              12.60                                                                              12.60                                                                              12.60                                                                              12.60                                                                              12.60                                                                              12.60                                          sitol                                                                         Nia-  2.02 2.02 2.02 2.02 2.02 2.02                                           cin-                                                                          amide                                                                         Pyrid-                                                                              2.00 --   2.00 --   2.00 2.00                                           oxal                                                                          .HCl                                                                          Pyrid-                                                                              0.031                                                                              2.031                                                                              0.031                                                                              2.031                                                                              0.031                                                                              0.031                                          oxine                                                                         .HCl                                                                          Ribo- 0.219                                                                              0.219                                                                              0.219                                                                              0.219                                                                              0.219                                                                              0.219                                          flavin                                                                        Thi-  2.17 2.17 2.17 2.17 2.17 2.17                                           amine                                                                         .HCl                                                                          Thy-  0.365                                                                              0.365                                                                              0.365                                                                              0.365                                                                              0.365                                                                              0.365                                          midine                                                                        Vi-   0.68 0.68 0.68 0.68 0.68 0.68                                           tamin                                                                         B.sub.12                                                                      __________________________________________________________________________

The preferred hypertrophy assay comprises:

(a) precoating the wells of 96-well tissue culture plates with a mediumcontaining calf serum, preferably D-MEM/F-12 medium containing 4% fetalcalf serum, wherein preferably the wells are incubated with the mediumfor about eight hours at about 37° C.;

(b) removing the medium;

(c) plating a suspension of myocytes in the inner 60 wells at 7.5×10⁴cells per mL in D-MEM/F-12 medium supplemented with insulin,transferrin, and aprotinin;

(d) culturing the myocytes for at least 24 hours;

(e) adding the test substance;

(f) culturing the cells with the test substance (preferably for about24-72 hours, more preferably for about 48 hours); and

(g) measuring for hypertrophy, preferably with crystal violet stain.

Preferably the medium used in step (c) is a serum-free medium alsocontaining penicillin/streptomycin (pen/strep) and glutamine. Mostpreferably, the medium contains 100 mL D-MEM/F-12, 100 μL transferrin(10 mg/mL), 20 μL insulin (5 mg/mL), 50 μL aprotinin (2 mg/mL), 1 mLpen/strep (JRH Biosciences No. 59602-77P), and 1 mL L-glutamine (200mM).

The assay capacity of 1000 single samples a week coupled with the smallsample size requirement of 100 μL or less has enabled an expressioncloning and protein purification that would have been impossible toaccomplish using the current methods available.

Another method for assaying hypertrophy involves measuring for atrialnatriuretic peptide (ANP) release by means of an assay that determinesthe competition for binding of ¹²⁵ I-rat ANP for a rat ANP receptorA-IgG fusion protein. The method suitable for use is similar to thatused for determining gp120 using a CD4-IgG fusion protein described byChamow et al., Biochemistry, 29: 9885-9891 (1990).

K. Neurotrophic Assay

The assay used for ciliary ganglion neurotrophic activity described inLeung, Neuron, 8: 1045-1053 (1992) is suitable herein. Briefly, ciliaryganglia are dissected from E7-E8 chick embryos and dissociated intrypsin-EDTA (Gibco 15400-013) diluted ten fold in phosphate-bufferedsaline for 15 minutes at 37° C. The ganglia are washed free of trypsinwith three washes of growth medium (high glucose D-MEM supplemented with10% fetal bovine serum, 1.5 mM glutamine, 100 μg/mL penicillin, and 100μg/mL strepomycin), and then gently triturated in 1 mL of growth mediuminto a single-cell suspension. Neurons are enriched by plating this cellmixture in 5 mL of growth media onto a 100-mm tissue culture dish for 4hours at 37° C. in a tissue culture incubator. During this time thenon-neuronal cells preferentially stick to the dish and neurons can begently washed free at the end of the incubation. The enriched neuronsare then plated into a 96-well plate previously coated with collagen. Ineach well, 1000 to 2000 cells are plated, in a final volume of 100 to250 μL, with dilutions of the CHF to be tested. Following a 2-4-dayincubation at 37° C., the number of live cells is assessed by staininglive cells using the vital dye metallothionine (MTT). One-fifth of thevolume of 5 mg/mL MTT (Sigma M2128) is added to the wells. After a2-4-hour incubation at 37° C., live cells (filled with a dense purpleprecipitate) are counted by phase microscopy at 100X magnification.

3. Uses and Therapeutic Compositions and Administration of CHF

CHF is believed to find use as a drug for treatment of mammals (e.g.,animals or humans) in vivo having heart failure, arrhythmic or inotropicdisorders, and/or peripheral neuropathies and other neurologicaldisorders involving motor neurons or other neurons in which CNTF isactive.

For example, CHF may be useful in treating congestive heart failure incases where ACE inhibitors cannot be employed or are not as effective.CHF optionally is combined with or administered in concert with otheragents for treating congestive heart failure, including ACE inhibitors.

The effective amount of ACE inhibitor to be administered, if employed,will be at the physician's or veterinarian's discretion. Dosageadministration and adjustment is done to achieve optimal management ofcongestive heart failure and ideally takes into account use of diureticsor digitalis, and conditions such as hypotension and renal impairment.The dose will additionally depend on such factors as the type ofinhibitor used and the specific patient being treated. Typically theamount employed will be the same dose as that used if the ACE inhibitorwere to be administered without CHF.

Thus, for example, a test dose of enalapril is 5 mg, which is thenramped up to 10-20 mg per day, once a day, as the patient tolerates it.As another example, captopril is initially administered orally to humanpatients in a test dose of 6.25 mg and the dose is then escalated, asthe patient tolerates it, to 25 mg twice per day (BID) or three timesper day (TID) and may be titrated to 50 mg BID or TID. Tolerance levelis estimated by determining whether decrease in blood pressure isaccompanied by signs of hypotension. If indicated, the dose may beincreased up to 100 mg BID or TID. Captopril is produced foradministration as the active ingredient, in combination withhydrochlorothiazide, and as a pH stabilized core having an enteric ordelayed release coating which protects captopril until it reaches thecolon. Captopril is available for administration in tablet or capsuleform. A discussion of the dosage, administration, indications andcontraindications associated with captopril and other ACE inhibitors canbe found in the Physicians Desk Reference, Medical Economics DataProduction Co., Montvale, N.J. 2314-2320 (1994).

CHF is also potentially useful in the generation, maturation, andsurvival of oligodendrocytes in vitro for protection of oligodendrocytesagainst natural and tumor necrosis factor-induced death, in the survivaland differentiation of astrocytes and the induction of type-2 astrocytedevelopment, and in the stimulation of the recombinant production oflow-affinity nerve growth factor receptor and CD-4 by rat centralnervous system (CNS) microglia.

CHF is also potentially useful in having a trophic effect on denervatedskeletal muscle. In addition, it is expected to have the proliferativeresponses and binding properties of hematopoietic cells transfected withlow-affinity receptors for leukemia inhibitory factor, oncostatin M, andciliary neurotrophic factor, to regulate fibrinogen gene expression inhepatocytes by binding to the interleukin-6 receptor, to have trophicactions on murine embryonic carcinoma cells, to be an endogenouspyrogen, and to have a mitogenic effect on human IMR 32 neuroblastomacells.

In addition, CHF is expected to enhance the response to nerve growthfactor of cultured rat sympathetic neurons, to maintain motoneurons andtheir target muscles in developing rats, to induce motor neuronsprouting in vivo, to promote the survival of neonatal rat corticospinalneurons in vitro, to prevent degeneration of adult rat substantia nigradopaminergic neurons in vivo, to alter the threshold of hippocampalpyramidal neuron sensitivity to excitotoxin damage, to prevent neuronaldegeneration and promote low-affinity NGF receptor production in theadult rat CNS, and to enhance neuronal survival in embryonic rathippocampal cultures.

These activities translate into the treatment of all neurodegenerativediseases by CHF, including peripheral neuropathies (motor and sensory),ALS, Alzheimer's disease, Parkinson's disease, stroke, Huntington'sdisease, and ophthalmologic diseases, for example, those involving theretina.

CHF may also be useful as an adjunct treatment of neurological disorderstogether with such neurotrophic factors as, e.g., CNTF, NGF, BDNF, NT-3,NT-4, and NT-5.

The nucleic acid encoding the CHF may be used as a diagnostic fortissue-specific typing. For example, such procedures as in situhybridization, northern and Southern blotting, and PCR analysis may beused to determine whether DNA and/or RNA encoding CHF is present in thecell type(s) being evaluated.

Isolated CHF polypeptide may also be used in quantitative diagnosticassays as a standard or control against which samples containing unknownquantities of CHF may be prepared.

Therapeutic formulations of CHF for treating heart failure andneurological disorders are prepared for storage by mixing CHF having thedesired degree of purity with optional physiologically acceptablecarriers, excipients, or stabilizers (Remington's PharmaceuticalSciences, supra), in the form of lyophilized cake or aqueous solutions.Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics, or polyethylene glycol (PEG).

CHF to be used for in vivo administration must be sterile. This isreadily accomplished by filtration through sterile filtration membranes,prior to or following lyophilization and reconstitution. CHF ordinarilywill be stored in lyophilized form or in solution.

Therapeutic CHF compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of CHF or CHF antibody administration is in accord with knownmethods, e.g., injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, orintralesional routes, or by sustained-release systems as noted below.CHF is administered continuously by infusion or by bolus injection. CHFantibody is administered in the same fashion, or by administration intothe blood stream or lymph.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethylmethacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15: 167-277 [1981]and Langer, Chem. Tech., 12: 98-105 [1982] or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 [1983]), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S--S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release CHF compositions also include liposomally entrappedCHF. Liposomes containing CHF are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980);EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patentapplication 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP102,324. Ordinarily the liposomes are of the small (about 200-800Angstroms) unilamellar type in which the lipid content is greater thanabout 30 mol. % cholesterol, the selected proportion being adjusted forthe optimal CHF therapy.

An effective amount of CHF to be employed therapeutically will depend,for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.A typical daily dosage might range from about 1 μg/kg to up to 100 mg/kgof patient body weight or more per day, depending on the factorsmentioned above, preferably about 10 μg/kg/day to 10 mg/kg/day.Typically, the clinician will administer CHF until a dosage is reachedthat achieves the desired effect for treatment of the heart or neuraldysfunction. For example, the amount would be one which increasesventricular contractility and decreases peripheral vascular resistanceor ameliorates or treats conditions of similar importance in congestiveheart failure patients. The progress of this therapy is easily monitoredby conventional assays.

4. CHF Antibody Preparation

(i) Starting Materials and Methods

Immunoglobulins (Ig) and certain variants thereof are known and manyhave been prepared in recombinant cell culture. For example, see U.S.Pat. No. 4,745,055; EP 256,654; EP 120,694; EP 125,023; EP 255,694; EP266,663; WO 88/03559; Faulkner et al., Nature, 298: 286 (1982);Morrison, J. Immun., 123: 793 (1979); Koehler et al., Proc. Natl. Acad.Sci. USA, 77: 2197 (1980); Raso et al., Cancer Res., 41: 2073 (1981);Morrison et al., Ann. Rev. Immunol., 2: 239 (1984); Morrison, Science,229: 1202 (1985); and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984). Reassorted immunoglobulin chains are also known. See, forexample, U.S. Pat. No. 4,444,878; WO 88/03565; and EP 68,763 andreferences cited therein. The immunoglobulin moiety in the chimeras ofthe present invention may be obtained from IgG-1, IgG-2, IgG-3, or IgG-4subtypes, IgA, IgE, IgD, or IgM, but preferably from IgG-1 or IgG-3.

(ii) Polyclonal antibodies

Polyclonal antibodies to CHF polypeptides or CHF fragments are generallyraised in animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of CHF or CHF fragment and an adjuvant. It may be useful toconjugate CHF or a fragment containing the target amino acid sequence toa protein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹ N═C═NR,where R and R¹ are different alkyl groups.

Animals are immunized against the CHF polypeptide or CHF fragment,immunogenic conjugates, or derivatives by combining 1 mg or 1 μg of thepeptide or conjugate (for rabbits or mice, respectively) with 3 volumesof Freund's complete adjuvant and injecting the solution intradermallyat multiple sites. One month later the animals are boosted with 1/5 to1/10 the original amount of peptide or conjugate in Freund's completeadjuvant by subcutaneous injection at multiple sites. Seven to 14 dayslater the animals are bled and the serum is assayed for CHF or CHFfragment antibody titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of the same CHF orCHF fragment, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

(iii) Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier"monoclonal" indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the CHF monoclonal antibodies of the invention may be madeusing the hybridoma method first described by Kohler and Milstein,Nature, 256: 495 (1975), or may be made by recombinant DNA methods(Cabilly et al., supra).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the CSF or CSF fragment used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 [Academic Press, 1986]).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. U.S.A., and SP-2cells available from the American Type Culture Collection, Rockville,Md. U.S.A.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against CHF. Preferably,the binding specificity of monoclonal antibodies produced by hybridomacells is determined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbentassay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson and Pollard, Anal.Biochem., 107: 220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxyapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of DNA encoding the antibody includeSkerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992).

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (Morrison, et al., Proc. Nat. Acad.Sci., 81: 6851 [1984]), or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid"antibodies are prepared that have the binding specificity of an anti-CHFmonoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for a CHF andanother antigen-combining site having specificity for a differentantigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I; a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; radioactive isotopic labels, such as, e.g., ¹²⁵I, ³² P, ¹⁴ C, or ³ H; or an enzyme, such as alkaline phosphatase,beta-galactosidase, or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al., Nature, 144: 945 (1962); David et al., Biochemistry,13: 1014 (1974); Pain et al., J. Immunol. Meth., 40: 219 (1981); andNygren, J. Histochem. and Cytochem., 30: 407 (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard(which may be a CHF or an immunologically reactive portion thereof) tocompete with the test sample analyte (CHF) for binding with a limitedamount of antibody. The amount of CHF in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the protein(CHF) to be detected. In a sandwich assay, the test sample analyte isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the analyte, thus forming aninsoluble three-part complex. David and Greene, U.S. Pat. No. 4,376,110.The second antibody may itself be labeled with a detectable moiety(direct sandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example; one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme (e.g., horseradishperoxidase).

(iv) Humanized antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as "import" residues, whichare typically taken from an "import" variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature 321, 522-525 [1986]; Riechmann et al., Nature 332,323-327 [1988]; Verhoeyen et al., Science 239, 1534-1536 [1988]), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such "humanized" antibodiesare chimeric antibodies (Cabilly et al., supra), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called "best-fit" method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151: 2296 [1993]; Chothia and Lesk, J. Mol. Biol., 196: 901[1987]). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89: 4285 [1992]; Presta et al., J. Immnol., 151: 2623 [1993]).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

(v) Human antibodies

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor,J. Immunol. 133, 3001 (1984); Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86-95 (1991).

It is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature,362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7: 33 (1993).

Alternatively, the phage display technology (McCafferty et al., Nature,348: 552-553 [1990]) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimicks someof the properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology, 3: 564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352: 624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol., 222: 581-597 (1991), or Griffith et al., EMBO J.,12: 725-734 (1993).

In a natural immune response, antibody genes accumulate mutations at ahigh rate (somatic hypermutation). Some of the changes introduced willconfer higher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as "chain shuffling" (Marks et al.,Bio/Technol., 10: 779-783 [1992]). In this method, the affinity of"primary" human antibodies obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from unimmunized donors. This technique allows theproduction of antibodies and antibody fragments with affinities in thenM range. A strategy for making very large phage antibody repertoireshas been described by Waterhouse et al., Nucl. Acids Res., 21: 2265-2266(1993).

Gene shuffling can also be used to derive human antibodies from rodentantibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as "epitope imprinting", the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT WO 93/06213,published 1 Apr. 1993). Unlike traditional humanization of rodentantibodies by CDR grafting, this technique provides completely humanantibodies, which have no framework or CDR residues of rodent origin.

(vi) Bispecific antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is for aCHF, the other one is for any other antigen, and preferably for anotherligand that binds to a GH/cytokine receptor family member. For example,bispecific antibodies specifically binding a CHF and neurotrophicfactor, or two different types of CHF polypeptides are within the scopeof the present invention.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537-539 [1983]), Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of 10 different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule, which is usually done by affinitychromatography steps, is rather cumbersome, and the product yields arelow. Similar procedures are disclosed in WO 93/08829 published 13 May1993, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

According to a different and more preferred approach, antibody-variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant-domain sequences.The fusion preferably is with an immunoglobulin heavy-chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1),containing the site necessary for light-chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the production of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation.

For further details of generating bispecific antibodies, see, forexample, Suresh et al., Methods in Enzymology, 121: 210 (1986).

(vii) Heteroconjugate antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (WO 91/00360; WO 92/00373; and EP03089). Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

5. Uses of CHF Antibodies

CHF antibodies are useful in diagnostic assays for CHF, e.g., itsproduction in specific cells, tissues, or serum. The antibodies arelabeled in the same fashion as CHF described above and/or areimmobilized on an insoluble matrix. In one embodiment of areceptor-binding assay, an antibody composition that binds to all or aselected plurality of CHFs is immobilized on an insoluble matrix, thetest sample is contacted with the immobilized antibody composition toadsorb all CHFs, and then the immobilized CHFs are contacted with aplurality of antibodies specific for each CHF, each of the antibodiesbeing individually identifiable as specific for a predetermined CHF, asby unique labels such as discrete fluorophores or the like. Bydetermining the presence and/or amount of each unique label, therelative proportion and amount of each CHF can be determined.

The antibodies of this invention are also useful in passively immunizingpatients.

CHF antibodies also are useful for the affinity purification of CHF fromrecombinant cell culture or natural sources. CHF antibodies that do notdetectably cross-react with the rat CHF can purify CHF free from suchprotein.

Suitable diagnostic assays for CHF and its antibodies are well known perse. In addition to the bioassays described in the examples below whereinthe candidate CHF is tested to see if it has hypertrophic,anti-arrhythmic, inotropic, or neurotrophic activity, competitive,sandwich and steric inhibition immunoassay techniques are useful. Thecompetitive and sandwich methods employ a phase-separation step as anintegral part of the method, while steric inhibition assays areconducted in a single reaction mixture. Fundamentally, the sameprocedures are used for the assay of CHF and for substances that bindCHF, although certain methods will be favored depending upon themolecular weight of the substance being assayed. Therefore, thesubstance to be tested is referred to herein as an analyte, irrespectiveof its status otherwise as an antigen or antibody, and proteins thatbind to the analyte are denominated binding partners, whether they beantibodies, cell-surface receptors, or antigens.

Analytical methods for CHF or its antibodies all use one or more of thefollowing reagents: labeled analyte analogue, immobilized analyteanalogue, labeled binding partner, immobilized binding partner, andsteric conjugates. The labeled reagents also are known as "tracers."

The label used (and this is also useful to label CHF nucleic acid foruse as a probe) is any detectable functionality that does not interferewith the binding of analyte and its binding partner. Numerous labels areknown for use in immunoassay, examples including moieties that may bedetected directly, such as fluorochrome, chemiluminscent, andradioactive labels, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected. Examples of such labels includethe radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I; fluorophores suchas rare earth chelates or fluorescein and its derivatives; rhodamine andits derivatives; dansyl; umbelliferone; luciferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456);luciferin; 2,3-dihydrophthalazinediones; malate dehydrogenase; urease;peroxidase such as horseradish peroxidase (HRP); alkaline phosphatase;β-galactosidase; glucoamylase; lysozyme; saccharide oxidases, e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase; heterocyclic oxidases such as uricase and xanthineoxidase, coupled with an enzyme that employs hydrogen peroxide tooxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase; biotin/avidin; spin labels; bacteriophage labels;stable free radicals; and the like.

Those of ordinary skill in the art will know of other suitable labelsthat may be employed in accordance with the present invention. Thebinding of these labels to CHF, antibodies, or fragments thereof can beaccomplished using standard techniques commonly known to those ofordinary skill in the art. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the polypeptide with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090(enzymes); Hunter et al., Nature, 144: 945 (1962); David et al.,Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol. Methods,40: 219-230 (1981); Nygren, J. Histochem and Cytochem., 30: 407-412(1982); O'Sullivan et al., "Methods for the Preparation ofEnzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods inEnzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (AcademicPress, New York, New York, 1981), pp. 147-166; Kennedy et al., Clin.Chim. Acta, 70: 1-31 (1976); and Schurs et al., Clin. Chim. Acta, 81:1-40 (1977). Coupling techniques mentioned in the lattermost referenceare the glutaraldehyde method, the periodate method, the dimaleimidemethod, and the m-maleimidobenzyl-N-hydroxysuccinimide ester method.

In the practice of the present invention, enzyme labels are a preferredembodiment. No single enzyme is ideal for use as a label in everyconceivable assay. Instead, one must determine which enzyme is suitablefor a particular assay system. Criteria important for the choice ofenzymes are turnover number of the pure enzyme (the number of substratemolecules converted to product per enzyme site per unit of time), purityof the enzyme preparation, sensitivity of detection of its product, easeand speed of detection of the enzyme reaction, absence of interferingfactors or of enzyme-like activity in the test fluid, stability of theenzyme and its conjugate, availability and cost of the enzyme and itsconjugate, and the like. Included among the enzymes used as preferredlabels in the assays of the present invention are alkaline phosphatase,HRP, beta-galactosidase, urease, glucose oxidase, glucoamylase, malatedehydrogenase, and glucose-6phosphate dehydrogenase. Urease is among themore preferred enzyme labels, particularly because of chromogenic pHindicators that make its activity readily visible to the naked eye.

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the binding partner from any analytethat remains free in solution. This conventionally is accomplished byeither insolubilizing the binding partner or analyte analogue before theassay procedure, as by adsorption to a water-insoluble matrix or surface(Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling (forexample, using glutaraldehyde cross-linking), or by insolubilizing thepartner or analogue afterward, e.g., by immunoprecipitation.

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer analogue to competewith the test sample analyte for a limited number of binding sites on acommon binding partner. The binding partner generally is insolubilizedbefore or after the competition and then the tracer and analyte bound tothe binding partner are separated from the unbound tracer and analyte.This separation is accomplished by decanting (where the binding partnerwas preinsolubilized) or by centrifuging (where the binding partner wasprecipitated after the competitive reaction). The amount of test sampleanalyte is inversely proportional to the amount of bound tracer asmeasured by the amount of marker substance. Dose-response curves withknown amounts of analyte are prepared and compared with the test resultsto quantitatively determine the amount of analyte present in the testsample. These assays are called ELISA systems when enzymes are used asthe detectable markers.

Another species of competitive assay, called a "homogeneous" assay, doesnot require a phase separation. Here, a conjugate of an enzyme with theanalyte is prepared and used such that when anti-analyte binds to theanalyte, the presence of the anti-analyte modifies the enzyme activity.In this case, CHF or its immunologically active fragments are conjugatedwith a bifunctional organic bridge to an enzyme such as peroxidase.Conjugates are selected for use with anti-CHF so that binding of theanti-CHF inhibits or potentiates the enzyme activity of the label. Thismethod per se is widely practiced under the name of EMIT.

Steric conjugates are used in steric hindrance methods for homogeneousassay. These conjugates are synthesized by covalently linking alow-molecular-weight hapten to a small analyte so that antibody tohapten substantially is unable to bind the conjugate at the same time asanti-analyte. Under this assay procedure the analyte present in the testsample will bind anti-analyte, thereby allowing anti-hapten to bind theconjugate, resulting in a change in the character of the conjugatehapten, e.g., a change in fluorescence when the hapten is a fluorophore.

Sandwich assays particularly are useful for the determination of CHF orCHF antibodies. In sequential sandwich assays an immobilized bindingpartner is used to adsorb test sample analyte, the test sample isremoved as by washing, the bound analyte is used to adsorb labeledbinding partner, and bound material is then separated from residualtracer. The amount of bound tracer is directly proportional to testsample analyte. In "simultaneous" sandwich assays the test sample is notseparated before adding the labeled binding partner. A sequentialsandwich assay using an anti-CHF monoclonal antibody as one antibody anda polyclonal anti-CHF antibody as the other is useful in testing samplesfor CHF activity.

The foregoing are merely exemplary diagnostic assays for CHF andantibodies. Other methods now or hereafter developed for thedetermination of these analytes are included within the scope hereof,including the bioassays described above.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all citations in the specification areexpressly incorporated herein by reference.

EXAMPLE I Identification and In Vitro Activity of a CHF

A. Assay for Expression-Cloned Material

The assay used for hypertrophy is an in vitro neonatal rat hearthypertrophy assay described in general as follows:

1. Preparation of the Myocyte Cell Suspension

The preparation of the myocyte cell suspension is based on methodsoutlined in Chien et al., J. Clin. Invest., 75: 1770-1780 (1985) andIwaki et al., supra. Ventricles from the hearts of 1-2 daySprague-Dawley rat pups were removed and trisected. The mincedventricles were digested with a series of sequential collagenasetreatments. Purification of the resulting single-cell suspension on adiscontinuous Percoll gradient resulted in a suspension of 95% puremyocytes.

2. Plating and Culture of the Myocytes

Two published methods for plating and culturing the myocytes are asfollows: (1) Long et al., supra, preplated the cell suspension for 30min. in MEM/5% calf serum. The unattached myocytes were then plated inserum-free MEM supplemented with insulin, transferrin, BrdU, and bovineserum albumin in 35-mm tissue-culture dishes at a density of 7.5×10⁴cells per mL. (2) Iwaki et al., supra, plated the cell suspension inD-MEM/199/5% horse serum/5% fetal calf serum in 10-cm tissue-culturedishes at 3×10⁵ cells per mL. After 24 hr in culture the cells werewashed and incubated in serum-free D-MEM/199.

A different protocol has been developed in accordance with thisinvention for plating and culturing these cells to increase testingcapacity with a miniaturized assay. The wells of 96-well tissue-cultureplates are precoated with D-MEM/F12/4% fetal calf serum for 8 hr at 37°C. This medium is removed and the cell suspension is plated in the inner60 wells at 7.5×10⁴ cells per mL in D-MEM/F-12 supplemented withinsulin, transferrin, and aprotinin. The medium typically also containsan antibiotic such as penicillin/streptomycin and glutamine. This mediumallows these cells to survive at this low plating density without serum.Test substances are added directly into the wells after the cells havebeen in culture for 24 hours.

3. Readout of Hypertrophy

After stimulation with alpha adrenergic agonists or endothelin, neonatalrat myocardial cells in culture display several features of the in vivocardiac muscle cell hypertrophy seen in congestive heart failure,including an increase in cell size and an increase in the assembly of anindividual contractile protein into organized contractile units. Chienet al., FASEB J., supra. These changes can be viewed with an invertedphase microscope and the degree of hypertrophy scored with an arbitraryscale of 7 to 0, with 7 being fully hypertrophied cells and 3 beingnon-stimulated cells. The 3 and 7 states may be seen in Simpson et al.,Circulation Research, 51: 787-801 (1982), FIG. 2, A and B, respectively.To facilitate the microscopic readout of the 96-well cultures and togenerate a permanent record, the myocytes are fixed and stained afterthe appropriate testing period with crystal violet stain in methanol.Crystal violet is a commonly used protein stain for cultured cells.

Additionally, an aliquot can be taken from the 96-well plates andmonitored for the expression of protein markers of the response such asrelease of ANF or ANP.

B. Expression Cloning

Poly(A)⁺ RNA was isolated (Aviv and Leder, Proc. Natl. Acad. Sci. USA,69: 1408-1412 [1972]; Cathala et al., DNA, 2: 329-335 [1983]) from day 7mouse embryoid bodies. Embryoid bodies were generated by thedifferentiation of pluripotent embryonic stem (ES) cells (Doetschman etal., J. Embryol. Exp. Morphol., 87: 27-45 [1985]). The embryonic stemcell line ES-D3 (ATCC No. CRL 1934) was maintained in anundifferentiated state in a medium containing LIF (Williams et al.,Nature, 336: 684-687 [1988]). This medium contained D-MEM (highglucose), 1% glutamine, 0.1 mM 2-mercaptoethanol,penicillin-streptomycin, 15% heat-inactivated fetal bovine serum, and 15ng/mL mouse LIF. When these cells were put into suspension culture inthe same medium without LIF and containing 20% heat-inactivated fetalbovine serum (day 0), they aggregated and differentiated intomulticellular structures called embryoid bodies. By day 8 of culture,beating primordial heart-like structures formed on a fraction of thebodies. The embryoid bodies were evaluated for the production of CHFactivity by changing the differentiating ES cells to serum-free medium(D-MEM/F-12, 1% glutamine, penicillin-streptomycin, containing 0.03%bovine serum albumin) for a 24-hour accumulation. Prior to assay, theconditioned medium was concentrated 10 fold with a 3-K ultrafiltrationmembrane (Amicon), and dialyzed against assay medium. Medium conditionedfor 24 hours starting at day 3 gave a hypertrophy score of 4.5-5.5, andstarting at day 6 a score of 5.5-7.5.

A cDNA library in the plasmid expression vector, pRK5B (Holmes et al.,Science, 253: 1278-1280 [1991]), was prepared following a vector primingstrategy (Strathdee et al., Nature, 356: 763-767 [1992]). The vector,pRK5B, was linearized at the NotI site, treated with alkalinephosphatase, and ligated to the single-stranded oligonucleotide,ocdl.1.3, having the sequence:

    5'-GCGGCCGCGAGCTCGAATTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

(SEQ ID NO: 5). The ligated product was then cut with BstXI, and the4700-bp vector fragment was isolated by agarose gel electrophoresis. Thevector was further purified by oligo dA chromatography.

The expression library was constructed using 1 μg of the poly (A)⁺ RNA,5 μg of vector primer, and reagents from Amersham. Following first- andsecond-strand synthesis and T4 DNA polymerase fill-in reactions, thematerial was sized for inserts of greater than 500 bp by gelelectrophoresis and circularized by blunt-end ligation without theaddition of linkers. The ligations were used to transform E. coli strainDH5α by electroporation. From 1 μg of poly(A)⁺ RNA, 499 ng ofdouble-stranded cDNA were generated. Seventeen nanograms of cDNA wereligated, and 3.3 ng were transformed to yield 780,000 clones, 83% ofwhich had inserts with an average size of 1470 bp.

DNA was isolated from pools of 75-15,000 clones and transfected intohuman embryonic kidney 293 cells by Lipofectamine transfection (GibcoBRL). Two micrograms of DNA were used to transfect ˜200,000 cells in6-well dishes. The cells were incubated in 2 mL of serum-free assaymedium for four days. This medium consisted of 100 mL D-MEM/F-12, 100 μLtransferrin (10 mg/mL), 20 μL insulin (5 mg/mL), 50 μL aprotinin (2mg/mL), 1 mL pen/strep (JRH Biosciences No. 59602-77P), and 1 mLL-glutamine (200 mM). Transfection and expression efficiency wasmonitored by the inclusion of 0.2 μg of DNA for a plasmid expressing asecreted form of alkaline phosphatase (Tate et al., FASEB J., 4: 227-231[1990]).

One hundred microliters of conditioned culture medium from eachtransfected pool was assayed for hypertrophy in a final volume of 200μL. For some pools the conditioned medium was concentrated 4-5 foldbefore assay with Centricon 3™ microconcentrators (Amicon). Ninety poolsof 10,000-15,000 clones, 359 pools of 1000-5800 clones, and 723 pools of75-700 clones were transfected and assayed for hypertrophy activity. Ofthese 1172 pools, two were found to be positive. Pool 437 (a pool of 187clones) and pool 781 (a pool of 700 clones) gave scores of 4. A pureclone (designated pchf.437.48) from pool 437 was isolated byretransfection of positive pools containing fewer and fewer numbers ofclones until a single clone was obtained. A pure clone from pool 781(designated pchf.781) was isolated by colony hybridization to the insertfrom clone pchf.437.48.

The sequence for the insert of clone pchf.781 is provided in FIG. 1 (SEQID NOS: 1, 2, and 3 for the two nucleotide strands and amino acidsequence, respectively). The sequence of the insert of clone pchf.437.48matches clone 781 starting at base 27 (underlined).

The first open reading frame of clone pchf.781 (see translation, FIG. 1)encodes a protein of 203 amino acids (translated MW=21.5 kDa). Thisprotein contains one cysteine residue, one potential N-linkedglycosylation site, and no hydrophobic N-terminal secretion signalsequence. The 3' untranslated region of clone pchf.781 contains a commonmouse repeat known as b1 (bp ˜895-1015). Hybridization of 7-day embryoidbody poly(A)⁺ RNA with a probe from clone pchf.781 shows a single bandof ˜1.4 kb, which is about the same size as the insert from the cDNAclones.

The encoded sequence is not highly similar (>35% amino acid identity) toany known protein sequences in the Dayhoff database. It does, however,show a low degree of similarity to a family of distantly relatedproteins including CNTF, interleukin-6 (IL-6), interleukin-11 (IL-11),LIF, and oncostatin M (OSM) (Bazan, Neuron, 7: 197-208 [1991]). MouseCHF has 24% amino acid identity with mouse LIF (Rose and Todaro, WO93/05169) and 21% amino acid identity with human CNTF (McDonald et al.,Biochim. Biophys. Acta, 1090: 70-80 [1991]). See FIG. 2 for an alignmentof mouse CHF and human CNTF sequences. CNTF, IL-6, IL-11, LIF, and OSMuse related receptor signaling proteins including gp130 that are membersof the GH/cytokine receptor family (Kishimoto et al., Cell, 76: 253-262[1994]). CNTF, like CHF, lacks an N-terminal secretion signal sequence.

C. Identity and Activity of Clone

To demonstrate that clone pchf.781 encodes a CHF, expression studieswere performed both by transfection of 293 cells and by utilizing acoupled in vitro SP6 transcription/translation system. ³⁵ S-methionineand cysteine labeling of the proteins produced by pchf.781-transfected293 cells (in comparison with vector-transfected cells) showed that theconditioned medium contained a labeled protein of about 21.8 kDa, andthat the cell extract showed a protein of 22.5 kDa. Conditioned mediafrom these transfections gave a morphology score of 6 when assayed forcardiac hypertrophy at a dilution of 1:4 using the assay describedabove. Conditioned media from unlabeled transfections gave a morphologyscore of 5.5-6.5 at a dilution of 1:1.

These assays were also positive for a second measure of cardiachypertrophy-ANP release. See FIG. 3. This assay was performed bydetermination of the competition for the binding of ¹²⁵ I-rat ANP for arat ANP receptor A-IgG fusion protein. This method is similar to thatused for the determination of gp120 using a CD4-IgG fusion protein(Chamow et al., Biochemistry, 29: 9885-9891 [1990]). Briefly, microtiterwells were coated with 100 μL of rat anti-human IgG antibody (2 μg/mL)overnight at 4° C. After washing with phosphate-buffered salinecontaining 0.5% bovine serum albumin, the wells were incubated with 100μL of 3 ng/mL rat ANP receptor A-IgG (produced and purified in a manneranalogous to the human ANP receptor A-IgG (Bennett et al., J. Biol.Chem., 266: 23060-23067 [1991]) for one hour at 24° C. The wells werewashed and incubated with 50 μL of rat ANP standard or sample for onehour at 24° C. Then 50 μL of ¹²⁵ I-rat ANP (Amersham) was added for anadditional one-hour incubation, The wells were washed and counted todetermine the extent of binding competition. ANP concentrations in thesamples were determined by comparison to a rat ANP standard curve.

³⁵ S-methionine labeling of the proteins made by SP6-coupled in vitrotranscription/translation (materials from Promega) of clone pchf.781showed a labeled protein of 22.4 kDa. The labeled translation productwas active when assayed for cardiac hypertrophy at a dilution of 1:200(morphology score 5-6). To verify that the 22.4-kDa-labeled band wasresponsible for the hypertrophy activity, the labeled translationproduct was applied to a reverse-phase C4 column (Synchropak RO-4-4000)equilibrated in 10% acetonitrile, 0.1% TFA, and eluted with anacetonitrile gradient. Coincident peaks of labeled protein andhypertrophy activity eluted from this column at ˜55% acetonitrile.

A cardiac myocyte hypertrophy activity has been reported and partiallypurified from rat cardiac fibroblasts. Long et al., supra. Toinvestigate further the identity of the CHF herein, rat cardiacfibroblasts were cultured. Conditioned medium from these primarycultures does have cardiac hypertrophy in the in vitro neonatal ratheart hypertrophy assay herein. Blot hybridization of rat fibroblastmRNA isolated from these cultures shows a clear band of 1.4 kb whenprobed with a coding region fragment of clone pchf.781. (Hybridizationwas performed in 5 x SSC, 20% formamide at 42° C. with a final wash in0.2 x SSC at 50° C.)

D. Purification of Factor

The culture medium conditioned by cells transfected with clone pchf.781or a human clone is adjusted to 1.5M NaCl and applied to a Toyopearl™Butyl-650M column. The column is washed with 10 mM TRIS-HCl, pH 7.5, 1MNaCl, and the activity eluted with 10 mM TRIS-HCl, pH 7.5, 10 mMZwittergent™ 3-10. The peak of activity is adjusted to 150 mM NaCl, pH8.0, and applied to a MONO-Q Fast Flow column. The column is washed with10 mM TRIS-HCl, pH 8.0, 150 mM NaCl, 0.1% octyl glucoside and activityis found in the flow-through fraction. The active material is thenapplied to a reverse phase C4 column in 0.1% TFA, 10% acetonitrile, andeluted with a gradient of 0.1% TFA up to 80%. The activity fractionatesat about 15-30 kDa on gel-filtration columns. It is expected that achaotrope such as guanidine-HCl is required for resolution and recovery.

EXAMPLE II Testing for in vivo Hypertrophy Activity

A. Normal Rats

The purified CHF from Example I is tested in normal rats to observe itseffect on cardiovascular parameters such as blood pressure, heart rate,systemic vascular resistance, contractility, force of heart beat,concentric or dilated hypertrophy, left ventricular systolic pressure,left ventricular mean pressure, left ventricular end-diastolic pressure,cardiac output, stroke index, histological parameters, ventricular size,wall thickness, etc.

B. Pressure-Overload Mouse Model

The purified CHF is also tested in the pressure-overload mouse modelwherein the pulmonary artery is constricted, resulting in rightventricular failure.

C. RV Murine Dysfunctional Model

A retroviral murine model of ventricular dysfunction can be used to testthe purified CHF, and the dP/dt, ejection fraction, and volumes can beassayed with the hypertrophy assay described above. In this model, thepulmonary artery of the mouse is constricted so as to generate pulmonaryhypertrophy and failure.

D. Transgenic Mouse Model

Transgenic mice that harbor a muscle actin promoter-IGF-I fusion genedisplay cardiac and skeletal muscle hypertrophy, without evidence ofmyopathy or heart failure. Further, IGF-I-gene-targeted mice displaydefects in cardiac myogenesis (as well as skeletal) including markedlydecreased expression of ventricular muscle contractile protein genes.The purified CHF is tested in these two models.

Additional genetic-based models of dilated cardiomyopathy and cardiacdysfunction, without necrosis, can be developed in transgenic andgene-targeted mice (MLC-ras mice; aortic banding of heterozygousIGF-I-deficient mice).

E. Post-Myocardial Infarction Rat Model

The purified CHF is also tested in a post-myocardial infarction ratmodel, which is predictive of human congestive heart failure inproducing natriuretic peptide. Specifically, male Sprague-Dawley rats(Charles River Breeding Laboratories, Inc., eight weeks of age) areacclimated to the facility for at least one week before surgery. Ratsare fed a pelleted rat chow and water ad libitum and housed in a light-and temperature-controlled room.

1. Coronary Arterial Ligation

Myocardial infarction is produced by left coronary arterial ligation asdescribed by Greenen et al., J. Appl. Physiol., 93: 92-96 (1987) andButtrick et al., Am. J. Physiol., 260: 11473-11479 (1991). The rats areanesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally),intubated via tracheotomy, and ventilated by a respirator (HarvardApparatus Model 683). After a left-sided thoracotomy, the left coronaryartery is ligated approximately 2 mm from its origin with a 7-0 silksuture. Sham animals undergo the same procedure except that the sutureis passed under the coronary artery and then removed. All rats arehandled according to the "Position of the American Heart Association onResearch Animal Use" adopted 11 Nov. 1984 by the American HeartAssociation. Four to six weeks after ligation, myocardial infarctioncould develop into heart failure in rats.

In clinical patients, myocardial infarction or coronary artery diseaseis the most common cause of heart failure. Congestive heart failure inthis model reasonably mimics congestive heart failure in most humanpatients.

2. Electrocardiograms

One week after surgery, electrocardiograms are obtained under lightmetofane anesthesia to document the development of infarcts. The ligatedrats of this study are subgrouped according to the depth and persistenceof pathological Q waves across the precordial leads. Buttrick et al.,supra; Kloner et al., Am. Heart J., 51: 1009-1013 (1983). This providesa gross estimate of infarct size and assures that large and smallinfarcts are not differently distributed in the ligated rats treatedwith CHF or CHF antagonist and vehicle. Confirmation is made by preciseinfarct size measurement.

3. CHF or CHF Antagonist Administration

Four weeks after surgery, CHF or CHF antagonist (10 μg/kg to 10 mg/kgtwice a day for 15 days) or saline vehicle is injected subcutaneously inboth ligated rats and sham controls. Body weight is measured twice aweek during the treatment. CHF or CHF antagonist is administered insaline or water as a vehicle.

4. Catheterization

After 13-day treatment with CHF, CHF antagonist, or vehicle, rats areanesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally). Acatheter (PE 10 fused with PE 50) filled with heparin-saline solution(50/U/mL) is implanted into the abdominal aorta through the rightfemoral artery for measurement of arterial pressure and heart rate. Asecond catheter (PE 50) is implanted into the right atrium through theright jugular vein for measurement of right atrial pressure and forsaline injection. For measurement of left ventricular pressures andcontractility (dP/dt), a third catheter (PE 50) is implanted into theleft ventricle through the right carotid artery. For the measurement ofcardiac output by a thermodilution method, a thermistor catheter (LyonsMedical Instrument Co., Sylmar, Calif.) is inserted into the aorticarch. The catheters are exteriorized at the back of the neck with theaid of a stainless-steel wire tunneled subcutaneously and then fixed.Following catheter implantation, all rats are housed individually.

5. Hemodynamic Measurements

One day after catherization, the thermistor catheter is processed in amicrocomputer system (Lyons Medical Instrument Co.) for cardiac outputdetermination, and the other three catheters are connected to a ModelCP-10 pressure transducer (Century Technology Company, Inglewood,Calif.) coupled to a Grass Model 7 polygraph (Grass Instruments, Quincy,Mass.). Mean arterial pressure (MAP), systolic arterial pressure (SAP),heart rate (HR), right atrial pressure (RAP), left ventricular systolicpressure (LVSP), left ventricular mean pressure (LVMP), left ventricularend-diastolic pressure (LVEDP), and left ventricular maximum (dP/dt) aremeasured in conscious, unrestrained rats.

For measurement of cardiac output, 0.1 mL of isotonic saline at roomtemperature is injected as a bolus via the jugular vein catheter. Thethermodilution curve is monitored by VR-16 simultrace recorders(Honeywell Co., N.Y.) and cardiac output (CO) is digitally obtained bythe microcomputer. Stroke volume (SV)=CO/HR; Cardiac index (CI)=CO/BW;Systemic vascular resistance (SVR)=MAP/CI.

After measurement of these hemodynamic parameters, 1 mL of blood iscollected through the arterial catheter. Serum is separated and storedat -70° C. for measurement of CHF levels or various biochemicalparameters if desired.

At the conclusion of the experiments, the rats are anesthetized withpentobarbital sodium (60 mg/kg) and the heart is arrested in diastolewith intra-atrial injection of KCl (1M). The heart is removed, and theatria and great vessels are trimmed from the ventricle. The ventricle isweighed and fixed in 10% buffered formalin.

All experimental procedures are approved by the Institutional AnimalCare and Use Committee of Genentech, Inc. before initiation of thestudy.

6. Infarct Size Measurements

The right ventricular free wall is dissected from the left ventricle.The left ventricle is cut in four transverse slices from apex to base.Five micrometer sections are cut and stained with Massons' trichromestain and mounted. The endocardial and epicardial circumferences of theinfarcted and non-infarcted left ventricle are determined with aplanimeter Digital Image Analyzer. The infarcted circumference and theleft ventricular circumference of all four slices are summed separatelyfor each of the epicardial and endocardial surfaces and the sums areexpressed as a ratio of infarcted circumference to left ventricularcircumference for each surface. These two ratios are then averaged andexpressed as a percentage for infarct size.

7. Statistical Analysis

Results are expressed as mean ±SEM. Two-way and one-way analysis ofvariance (ANOVA) is performed to assess differences in parameters amonggroups. Significant differences are then subjected to post hoc analysisusing the Newman-Keuls method. p<0.05 is considered significant.

8. Results

The mean body weight before and after treatment with CHF or CHFantagonist or vehicle is not expected to be different among theexperimental groups. Infarct size in ligated rats is not expected todiffer between the vehicle-treated group and the CHF- orCHF-antagonist-treated group.

It is expected that administration of CHF or CHF antagonist to theligated rats in the doses set forth above would result in improvedcardiac hypertrophy by increasing ventricular contractility anddecreasing peripheral vascular resistance over that observed with thevehicle-treated sham and ligated rat controls. This expected resultwould demonstrate that administration of CHF or CHF antagonist improvescardiac function in congestive heart failure. In sham rats, however, CHFor CHF antagonist administration at this dose is not expected to altersignificantly cardiac function except possibly slightly loweringarterial pressure and peripheral vascular resistance.

It would be reasonably expected that the rat data herein may beextrapolated to horses, cows, humans, and other mammals, correcting forthe body weight of the mammal in accordance with recognized veterinaryand clinical procedures. Using standard protocols and procedures, theveterinarian or clinician will be able to adjust the doses, scheduling,and mode of administration of CHF or a CHF antagonist to achieve maximaleffects in the desired mammal being treated. Humans are believed torespond in this manner as well.

EXAMPLE III Proposed Clinical Treatment of Dilated Cardiomyopathy

A. Intervention

Patient self-administration of CHF or CHF antagonist at an initial doseof 10-150 μg/kg/day is proposed. The dose would be adjusted downward foradverse effects. If no beneficial effects and no limiting adverseeffects are determined at the time of re-evaluation, the dose would beadjusted upward. Concurrent medication doses (e.g., captopril as an ACEinhibitor and diuretics) would be adjusted at the discretion of thestudy physician. After the maximum dose is administered for 8 weeks, theCHF or CHF antagonist administration is stopped, and re-evaluation isperformed after a similar time period off treatment (or a placebo).

B. Inclusion Criteria

Patients would be considered for the study if they meet the followingcriteria:

Dilated cardiomyopathy (DCM). Idiopathic DCM, or ischemic DCM withoutdiscrete areas of akinesis/dyskinesis of the left ventricle (LV) oncontrast ventriculography or 2D echocardiographyo Evidence for impairedsystolic function to include either LV end-diastolic dimension (EDD)>3.2cm/m² BSA or EDV>82 mL/m² on 2D echocardiography, LV fractionalshortening<28% on echocardiography, or ejection fraction (by contrastventriculography or radionuclide angiography)<0.49.

Symptoms. New York Heart Association class III or peak exercise VO₂ <16mL/kg/min. (adjusted for age), stable for at least one month on digoxin,diuretics, and vasodilators (ACE inhibitors).

Concurrent ACE inhibitor therapy.

Adequate echocardiographic "windows" to permit assessment of leftventricular volume and mass.

Ability to self-administer CHF or CHF antagonist according to the dosageschedule, and to return reliably for follow-up assessments.

Consent of patient and patient's primary physician to participate.

Absence of exclusion criteria.

C. Exclusion Criteria

Patients would be excluded from consideration for any of the followingreasons:

Dilated cardiomyopathy resulting from valvular heart disease (operableor not), specific treatable etiologies (including alcohol, if abstinencehas not been attempted), or operable coronary artery disease.

Exercise limited by chest pain or obstructive peripheral vasculardisease.

Chronic obstructive lung disease.

Diabetes mellitus or impaired glucose tolerance.

History of carpal tunnel syndrome, or evidence for positive Tinel's signon examination.

History of kidney stones.

Symptomatic osteoarthritis.

Inability to consent for or participate in serial bicycle ergometry withinvasive hemodynamic monitoring (as described below).

Active malignancy.

D. Patient Assessment

1) Major Assessment Points: baseline; after peak stable CHF or CHFantagonist dose maintained for 8 weeks; after equal period after drugdiscontinuation.

It is anticipated that patients would remain in the hospital for two tothree days at the onset of active treatment, with daily weights andlaboratory data including electrolytes, phosphorus, BUN, creatinine, andglucose. Following this, they would be monitored on the ClinicalResearch Center floor daily for an additional two to three days.

i. Physical examination.

ii. Symptom Point Score (Kelly et al., Amer. Heart J., 119: 1111[1990]).

iii. Laboratory data: CBC; electrolytes (including Mg⁺² and Ca⁺²); BUN;creatinine; phosphorus; fasting glucose and lipid profile (totalcholesterol, HDL-C, LDL-C, triglycerides); liver function tests (AST,ALT, alkaline phosphatase, total bilirubin); total protein; albumin;uric acid; and CHF.

iv. 2D, M-mode, and doppler echocardiography, including: diastolic andsystolic dimensions at the papillary muscle level; ejection fractionestimate by area planimetry from apical 2-chamber and 4-chamber views,estimated systolic and diastolic volumes by Simpson's rule method, andestimated left ventricular mass; doppler assessment of mitral valveinflow profile (IVRT, peak E, peak A, deceleration time, A waveduration), and pulmonary vein flow profile (systolic flow area,diastolic flow area, A reversal duration, and velocity).

v. Rest and exercise hemodynamics and measured oxygen consumption, usingbicycle ergometry with percutaneously inserted pulmonary artery andarterial catheters. Perceived exertion level would be scored on the Borgscale, and measurements of pulmonary artery systolic, diastolic, andmean pressures, as well as arterial pressures and pulmonary capillarywedge pressure would be measured at each increment of workload, alongwith arterial and mixed venous oxygen content for calculating cardiacoutput.

vi. Assessment of body fat and lean body mass, as well as skeletalmuscle strength and endurance.

2) Interim Assessment Points: weekly

i. Physical examination.

ii. Symptom Point Score.

iii. Laboratory data: electrolytes, BUN, creatinine, phosphorus, fastingglucose, somatomedin-C, and CHF.

E. Potential Benefits

1) Improved sense of well-being.

2) Increased exercise tolerance.

3) Increased muscle strength and lean body mass.

4) Decreased systemic vascular resistance.

5) Enhanced cardiac performance.

6) Enhanced compensatory myocardial hypertrophy.

EXAMPLE IV Testing for in vitro Neurotrophic Activity

An assay used for ciliary ganglion neurotrophic activity was performedas described in Leung, Neuron, 8: 1045-1053 (1992). Briefly, ciliaryganglia were dissected from E7-E8 chick embryos and dissociated intrypsin-EDTA (Gibco 15400-013) diluted ten fold in phosphate-bufferedsaline for 15 minutes at 37° C. The ganglia were washed free of trypsinwith three washes of growth medium (high glucose D-MEM supplemented with10% fetal bovine serum, 1.5 mM glutamine, 100 μg/mL penicillin, and 100μg/mL strepomycin), and then gently triturated in 1 mL of growth mediuminto a single-cell suspension. Neurons were enriched by plating thiscell mixture in 5 mL of growth media onto a 100-mm tissue culture dishfor 4 hours at 37° C. in a tissue culture incubator. During this timethe non-neuronal cells preferentially stuck to the dish and neurons weregently washed free at the end of the incubation.

The enriched neurons were then plated into a 96-well plate previouslycoated with collagen. In each well, 1000 to 2000 cells were plated, in afinal volume of 100 to 250 μL, with dilutions of the conditioned mediumfrom the pchf.781-transfected 293 cells of Example I. The cells werealso plated with the transfected 293 conditioned medium as a control,and with a CNTF standard as a comparison. Following a 2-4-day incubationat 37° C., the number of live cells was assessed by staining live cellsusing the vital dye metallothionine (MTT). One-fifth of the volume of 5mg/mL MTT (Sigma M2128) was added to the wells. After a 2-4-hourincubation at 37° C., live cells (filled with a dense purpleprecipitate) were counted by phase microscopy at 100X magnification.

The results of the assay are shown in FIG. 4. It can be seen that thepchf.781 transfection (triangles) increased survival of the live neurons(measured by cell count) as the fraction of assay volume of transfected293 conditioned medium increased. This is similar to the pattern for theCNTF standard (circles), and is in contrast to the control transfection(squares), which showed no increase in survival as a function ofincreased fraction of assay volume of conditioned medium. This indicatesthat CHF is useful as a neurotrophic agent, having a similar effect tothat observed with CNTF.

EXAMPLE V

A source of mRNA encoding human CHF (also known as human cardiotrophin-1[CT-1]) was identified by screening poly(A)+RNA from several adulttissues with a probe from the mouse CHF cDNA clones. Heart, skeletalmuscle, colon, ovary, and prostate showed a 1.8 kb band upon blothybridization with a 180-bp mouse CHF probe (extending from 19 bp 5' ofthe initiating ATG through amino acid 50) in 20% formamide, 5 X SSC at42° C. with a final wash at 0.25 X SSC at 52° C. Clones encoding humanCT-1 were isolated by screening a human heart cDNA library (Clontech)with the same probe and conditions (final wash at 55° C.).

Eleven clones were isolated from 1 million screened. The EcoRI insertsof several of the clones were subcloned into plasmid vectors and theirDNA sequences determined.

The DNA sequence from clone h5 (SEQ ID NOS: 6 and 7 for the sense andanti-sense strands, respectively) is shown in FIG. 5 and includes thewhole coding region. Clone h5 (pBSSK+.hu.CT1.h5) was deposited on Jul.26, 1994 in the American Type Culture Collection as ATCC No. 75,841. TheDNA sequence of another clone, designated h6, matches that of clone h5in the region of overlap. Clone h6 begins at base 47 of clone h5 andextends 3' of clone h5 for an additinal 521 bases. The encoded proteinsequence of human CT-1 (SEQ ID NO: 8) is 79% identical with the mouseCHF sequence (SEQ ID NO: 3), as evident from FIG. 6, wherein the formeris designated "humct1" and the latter is designated "chf.781."

To show that human CT-1 encoded by clone h5 is biologically active, theEcoRI fragment was cloned into the mammalian expression vector pRK5 (EP307,247) at the unique EcoRI site to give the plasmid pRK5.hu. CT1. Thisplasmid was transfected into human 293 cells, and the cells weremaintained in serum-free medium for 3-4 days. This medium was thenassayed for cardiac myocyte hypertrophy as described above for mouseCHF. The transfected 293 conditioned medium was clearly active in thisassay (hypertrophy score of 5.5 at a dilution of 1:20).

Deposit of Material

The following plasmid has been deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., U.S.A. (ATCC):

    ______________________________________                                        Plasmid        ATCC Dep. No.                                                                              Deposit Date                                      ______________________________________                                        pBSSK+.hu.CT1.h5                                                                             75,841       July 26, 1994                                     ______________________________________                                    

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe plasmid on deposit should die or be lost or destroyed whencultivated under suitable conditions, the plasmid will be promptlyreplaced on notification with another of the same plasmid. Availabilityof the deposited plasmid is not to be construed as a license to practicethe invention in contravention of the rights granted under the authorityof any government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1352 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGATAAGCCTGGGGCCAGCATGAGCCAGAGGGAGGGAAGTCTGGAAGACC50                          ACCAGACTGACTCCTCAATCTCATTCCTACCCCATTTGGAGGCCAAGATC100                         CGCCAGACACACAACCTTGCCCGCCTCCTGACCAAATATGCAGAACAACT150                         TCTGGAGGAATACGTGCAGCAACAGGGAGAGCCCTTTGGGCTGCCGGGCT200                         TCTCACCACCGCGGCTGCCGCTGGCCGGCCTGAGTGGCCCGGCTCCGAGC250                         CATGCAGGGCTACCGGTGTCCGAGCGGCTGCGGCAGGATGCAGCCGCCCT300                         GAGTGTGCTGCCCGCGCTGTTGGATGCCGTCCGCCGCCGCCAGGCGGAGC350                         TGAACCCGCGCGCCCCGCGCCTGCTGCGGAGCCTGGAGGACGCAGCCCGC400                         CAGGTTCGGGCCCTGGGCGCCGCGGTGGAGACAGTGCTGGCCGCGCTGGG450                         CGCTGCAGCCCGCGGGCCCGGGCCAGAGCCCGTCACCGTCGCCACCCTCT500                         TCACGGCCAACAGCACTGCAGGCATCTTCTCAGCCAAGGTGCTGGGGTTC550                         CACGTGTGCGGCCTCTATGGCGAGTGGGTGAGCCGCACAGAGGGCGACCT600                         GGGCCAGCTGGTGCCAGGGGGCGTCGCCTGAGAGTGAATACTTTTTCTTG650                         TAAGCTCGCTCTGTCTCGCCTCTTTGGCTTCAAATTTTCTGTCTCTCCAT700                         CTGTGTCCTGTGTGTTCTTGGGCTGTCCCTATCTTTCTGCATTTGTGTGG750                         TCTCTCTCTTCTGCTCTCCTCTCTGCAGGGAGCTTCTTTTTTCCAACAGT800                         TTCTCGTTTTGTCTCTCTCCAGTCTTGAACACTTTTGTCTCCGAGAGGTC850                         TCTTTTTGTTTCCTTGTCTCTTGGTTCTTTCTTTGCTTGCTTGCTTGCTT900                         GCTTGCTTGTTGTTGAGACAGGGTCTCACCATATAGCTCTGGATGGCCTG950                         GAACTTGCTATGTAGGCCAGGCTGGCCTCCAGCTCATAGAGATCCACTTG1000                        CCTCCGACTCCCAATTTCCCCATCTGTCTCCCTGTGATCCATATGGGTAT1050                        GTGTAACCCTTACTTTGTCTCATGGAGGTGACAATTTTTCTCCCTTCAGT1100                        TTCTTTGTTCTTTACTGACCAGAAAAGTGCCTACTTGTCCCCTGGTGGCA1150                        AGGCCATTCACCTTAGGACCTTCCCACCAGTTCCTTTGTAGGCAAATCCC1200                        TCCCCCTTTGAGGTCCTTCCCTTTCATACCGCCCTAGGCTGGTCAATGGA1250                        GAGAGAAAGGCAGAAAAACATCTTTAAAGAGTTTTATTTGAGAATAAATT1300                        AATTTTTGTAAATAAAATGTTTAACAATAAAACTAAACTTTTATGAAAAA1350                        AA1352                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1352 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CCTATTCGGACCCCGGTCGTACTCGGTCTCCCTCCCTTCAGACCTTCTGG50                          TGGTCTGACTGAGGAGTTAGAGTAAGGATGGGGTAAACCTCCGGTTCTAG100                         GCGGTCTGTGTGTTGGAACGGGCGGAGGACTGGTTTATACGTCTTGTTGA150                         AGACCTCCTTATGCACGTCGTTGTCCCTCTCGGGAAACCCGACGGCCCGA200                         AGAGTGGTGGCGCCGACGGCGACCGGCCGGACTCACCGGGCCGAGGCTCG250                         GTACGTCCCGATGGCCACAGGCTCGCCGACGCCGTCCTACGTCGGCGGGA300                         CTCACACGACGGGCGCGACAACCTACGGCAGGCGGCGGCGGTCCGCCTCG350                         ACTTGGGCGCGCGGGGCGCGGACGACGCCTCGGACCTCCTGCGTCGGGCG400                         GTCCAAGCCCGGGACCCGCGGCGCCACCTCTGTCACGACCGGCGCGACCC450                         GCGACGTCGGGCGCCCGGGCCCGGTCTCGGGCAGTGGCAGCGGTGGGAGA500                         AGTGCCGGTTGTCGTGACGTCCGTAGAAGAGTCGGTTCCACGACCCCAAG550                         GTGCACACGCCGGAGATACCGCTCACCCACTCGGCGTGTCTCCCGCTGGA600                         CCCGGTCGACCACGGTCCCCCGCAGCGGACTCTCACTTATGAAAAAGAAC650                         ATTCGAGCGAGACAGAGCGGAGAAACCGAAGTTTAAAAGACAGAGAGGTA700                         GACACAGGACACACAAGAACCCGACAGGGATAGAAAGACGTAAACACACC750                         AGAGAGAGAAGACGAGAGGAGAGACGTCCCTCGAAGAAAAAAGGTTGTCA800                         AAGAGCAAAACAGAGAGAGGTCAGAACTTGTGAAAACAGAGGCTCTCCAG850                         AGAAAAACAAAGGAACAGAGAACCAAGAAAGAAACGAACGAACGAACGAA900                         CGAACGAACAACAACTCTGTCCCAGAGTGGTATATCGAGACCTACCGGAC950                         CTTGAACGATACATCCGGTCCGACCGGAGGTCGAGTATCTCTAGGTGAAC1000                        GGAGGCTGAGGGTTAAAGGGGTAGACAGAGGGACACTAGGTATACCCATA1050                        CACATTGGGAATGAAACAGAGTACCTCCACTGTTAAAAAGAGGGAAGTCA1100                        AAGAAACAAGAAATGACTGGTCTTTTCACGGATGAACAGGGGACCACCGT1150                        TCCGGTAAGTGGAATCCTGGAAGGGTGGTCAAGGAAACATCCGTTTAGGG1200                        AGGGGGAAACTCCAGGAAGGGAAAGTATGGCGGGATCCGACCAGTTACCT1250                        CTCTCTTTCCGTCTTTTTGTAGAAATTTCTCAAAATAAACTCTTATTTAA1300                        TTAAAAACATTTATTTTACAAATTGTTATTTTGATTTGAAAATACTTTTT1350                        TT1352                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 203 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetSerGlnArgGluGlySerLeuGluAspHisGlnThrAspSer                                 151015                                                                        SerIleSerPheLeuProHisLeuGluAlaLysIleArgGlnThr                                 202530                                                                        HisAsnLeuAlaArgLeuLeuThrLysTyrAlaGluGlnLeuLeu                                 354045                                                                        GluGluTyrValGlnGlnGlnGlyGluProPheGlyLeuProGly                                 505560                                                                        PheSerProProArgLeuProLeuAlaGlyLeuSerGlyProAla                                 657075                                                                        ProSerHisAlaGlyLeuProValSerGluArgLeuArgGlnAsp                                 808590                                                                        AlaAlaAlaLeuSerValLeuProAlaLeuLeuAspAlaValArg                                 95100105                                                                      ArgArgGlnAlaGluLeuAsnProArgAlaProArgLeuLeuArg                                 110115120                                                                     SerLeuGluAspAlaAlaArgGlnValArgAlaLeuGlyAlaAla                                 125130135                                                                     ValGluThrValLeuAlaAlaLeuGlyAlaAlaAlaArgGlyPro                                 140145150                                                                     GlyProGluProValThrValAlaThrLeuPheThrAlaAsnSer                                 155160165                                                                     ThrAlaGlyIlePheSerAlaLysValLeuGlyPheHisValCys                                 170175180                                                                     GlyLeuTyrGlyGluTrpValSerArgThrGluGlyAspLeuGly                                 185190195                                                                     GlnLeuValProGlyGlyValAla                                                      200203                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 200 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAlaPheThrGluHisSerProLeuThrProHisArgArgAsp                                 151015                                                                        LeuCysSerArgSerIleTrpLeuAlaArgLysIleArgSerAsp                                 202530                                                                        LeuThrAlaLeuThrGluSerTyrValLysHisGlnGlyLeuAsn                                 354045                                                                        LysAsnIleAsnLeuAspSerAlaAspGlyMetProValAlaSer                                 505560                                                                        ThrAspGlnTrpSerGluLeuThrGluAlaGluArgLeuGlnGlu                                 657075                                                                        AsnLeuGlnAlaTyrArgThrPheHisValLeuLeuAlaArgLeu                                 808590                                                                        LeuGluAspGlnGlnValHisPheThrProThrGluGlyAspPhe                                 95100105                                                                      HisGlnAlaIleHisThrLeuLeuLeuGlnValAlaAlaPheAla                                 110115120                                                                     TyrGlnIleGluGluLeuMetIleLeuLeuGluTyrLysIlePro                                 125130135                                                                     ArgAsnGluAlaAspGlyMetProIleAsnValGlyAspGlyGly                                 140145150                                                                     LeuPheGluLysLysLeuTrpGlyLeuLysValLeuGlnGluLeu                                 155160165                                                                     SerGlnTrpThrValArgSerIleHisAspLeuArgPheIleSer                                 170175180                                                                     SerHisGlnThrGlyIleProAlaArgGlySerHisTyrIleAla                                 185190195                                                                     AsnAsnLysLysMet                                                               200                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCGGCCGCGAGCTCGAATTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT50                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1018 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GTGAAGGGAGCCGGGATCAGCCAGGGGCCAGCATGAGCCGGAGGGAGGGA50                          AGTCTGGAAGACCCCCAGACTGATTCCTCAGTCTCACTTCTTCCCCACTT100                         GGAGGCCAAGATCCGTCAGACACACAGCCTTGCGCACCTCCTCACCAAAT150                         ACGCTGAGCAGCTGCTCCAGGAATATGTGCAGCTCCAGGGAGACCCCTTC200                         GGGCTGCCCAGCTTCTCGCCGCCGCGGCTGCCGGTGGCCGGCCTGAGCGC250                         CCCGGCTCCGAGCCACGCGGGGCTGCCAGTGCACGAGCGGCTGCGGCTGG300                         ACGCGGCGGCGCTGGCCGCGCTGCCCCCGCTGCTGGACGCAGTGTGTCGC350                         CGCCAGGCCGAGCTGAACCCGCGCGCGCCGCGCCTGCTGCGCCGCCTGGA400                         GGACGCGGCGCGCCAGGCCCGGGCCCTGGGCGCCGCCGTGGAGGCCTTGC450                         TGGCCGCGCTGGGCGCCGCCAACCGCGGGCCCCGGGCCGAGCCCCCCGCC500                         GCCACCGCCTCAGCCGCCTCCGCCACCGGGGTCTTCCCCGCCAAGGTGCT550                         GGGGCTCCGCGTTTGCGGCCTCTACCGCGAGTGGCTGAGCCGCACCGAGG600                         GCGACCTGGGCCAGCTGCTGCCCGGGGGCTCGGCCTGAGCGCCGCGGGGC650                         AGCTCGCCCCGCCTCCTCCCGCTGGGTTCCGTCTCTCCTTCCGCTTCTTT700                         GTCTTTCTCTGCCGCTGTCGGTGTCTGTCTGTCTGCTCTTAGCTGTCTCC750                         ATTGCCTCGGCCTTCTTTGCTTTTTGTGGGGGAGAGGGGAGGGGACGGGC800                         AGGGTCTCTGTCGCCCAGGCTGGGGTGCAGTGGCGCGATCCCAGCACTGC850                         AGCCTCAACCTCCTGGGCTCAAGCCATCCTTCCGCCTCAGCTTCCCCAGC900                         AGCTGGGACTACAGGCACGCGCCACCACAGCCGGCTAATTTTTTATTTAA950                         TTTTTTGTAGAGACGAGGTTTCGCCATGTTGCCCAGGCTGGTCTTGAACT1000                        CCGGGGCTCAAGCGATCC1018                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1018 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CACTTCCCTCGGCCCTAGTCGGTCCCCGGTCGTACTCGGCCTCCCTCCCT50                          TCAGACCTTCTGGGGGTCTGACTAAGGAGTCAGAGTGAAGAAGGGGTGAA100                         CCTCCGGTTCTAGGCAGTCTGTGTGTCGGAACGCGTGGAGGAGTGGTTTA150                         TGCGACTCGTCGACGAGGTCCTTATACACGTCGAGGTCCCTCTGGGGAAG200                         CCCGACGGGTCGAAGAGCGGCGGCGCCGACGGCCACCGGCCGGACTCGCG250                         GGGCCGAGGCTCGGTGCGCCCCGACGGTCACGTGCTCGCCGACGCCGACC300                         TGCGCCGCCGCGACCGGCGCGACGGGGGCGACGACCTGCGTCACACAGCG350                         GCGGTCCGGCTCGACTTGGGCGCGCGCGGCGCGGACGACGCGGCGGACCT400                         CCTGCGCCGCGCGGTCCGGGCCCGGGACCCGCGGCGGCACCTCCGGAACG450                         ACCGGCGCGACCCGCGGCGGTTGGCGCCCGGGGCCCGGCTCGGGGGGCGG500                         CGGTGGCGGAGTCGGCGGAGGCGGTGGCCCCAGAAGGGGCGGTTCCACGA550                         CCCCGAGGCGCAAACGCCGGAGATGGCGCTCACCGACTCGGCGTGGCTCC600                         CGCTGGACCCGGTCGACGACGGGCCCCCGAGCCGGACTCGCGGCGCCCCG650                         TCGAGCGGGGCGGAGGAGGGCGACCCAAGGCAGAGAGGAAGGCGAAGAAA700                         CAGAAAGAGACGGCGACAGCCACAGACAGACAGACGAGAATCGACAGAGG750                         TAACGGAGCCGGAAGAAACGAAAAACACCCCCTCTCCCCTCCCCTGCCCG800                         TCCCAGAGACAGCGGGTCCGACCCCACGTCACCGCGCTAGGGTCGTGACG850                         TCGGAGTTGGAGGACCCGAGTTCGGTAGGAAGGCGGAGTCGAAGGGGTCG900                         TCGACCCTGATGTCCGTGCGCGGTGGTGTCGGCCGATTAAAAAATAAATT950                         AAAAAACATCTCTGCTCCAAAGCGGTACAACGGGTCCGACCAGAACTTGA1000                        GGCCCCGAGTTCGCTAGG1018                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 201 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetSerArgArgGluGlySerLeuGluAspProGlnThrAspSer                                 151015                                                                        SerValSerLeuLeuProHisLeuGluAlaLysIleArgGlnThr                                 202530                                                                        HisSerLeuAlaHisLeuLeuThrLysTyrAlaGluGlnLeuLeu                                 354045                                                                        GlnGluTyrValGlnLeuGlnGlyAspProPheGlyLeuProSer                                 505560                                                                        PheSerProProArgLeuProValAlaGlyLeuSerAlaProAla                                 657075                                                                        ProSerHisAlaGlyLeuProValHisGluArgLeuArgLeuAsp                                 808590                                                                        AlaAlaAlaLeuAlaAlaLeuProProLeuLeuAspAlaValCys                                 95100105                                                                      ArgArgGlnAlaGluLeuAsnProArgAlaProArgLeuLeuArg                                 110115120                                                                     ArgLeuGluAspAlaAlaArgGlnAlaArgAlaLeuGlyAlaAla                                 125130135                                                                     ValGluAlaLeuLeuAlaAlaLeuGlyAlaAlaAsnArgGlyPro                                 140145150                                                                     ArgAlaGluProProAlaAlaThrAlaSerAlaAlaSerAlaThr                                 155160165                                                                     GlyValPheProAlaLysValLeuGlyLeuArgValCysGlyLeu                                 170175180                                                                     TyrArgGluTrpLeuSerArgThrGluGlyAspLeuGlyGlnLeu                                 185190195                                                                     LeuProGlyGlySerAla                                                            200201                                                                        __________________________________________________________________________

What is claimed is:
 1. An isolated polypeptide having the translatedhuman cardiotrophin-1 (CT-1) amino acid sequence (SEQ ID NO: 8) shown inFIG.
 5. 2. The isolated polypeptide of claim 1 that has a molecularweight on reducing SDS-PAGE of about 21-23 kD.
 3. The polypeptide ofclaim 1, wherein a substituted conserved amino acid is located in aglycosylation site in the translated human cardiotrophin-1 (CT-1) aminoacid sequence (SEQ ID NO: 8) of FIG. 5.